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ACS Infectious Disease Award:
08:00am - 11:30am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Phoebe Glazer, Organizer; Ekaterina Pletneva, Organizer; Courtney Aldrich, Presider
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
Introductory Remarks
08:00am - 08:05am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

Sunday
3757682 - Avoiding Drug Resistance: Lessons from viral proteases, from HIV to SARS-CoV-2
08:05am - 09:00am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Celia Schiffer, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding, but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target’s function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. This rationale was derived from our lab’s experience with substrate recognition and drug resistance in the viral proteases of HIV, HCV and most recently applying to emerging target of SARS-CoV-2. This resulted in our development of the strategy of the substrate envelope and the use of parallel MD and machine learning to strategically avoid drug resistance and develop robust inhibitors.
Sunday
3731027 - A bacterial toxin inhibits translation in competitor cells
09:00am - 09:45am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
ADP-ribosyltransferase (ART) toxins were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they transfer an ADP-ribose moiety from NAD+ to an acceptor protein thereby inactivating key cellular processes and promoting bacterial pathogenesis. Since these early studies, our understanding of ART function has expanded beyond the ADP-ribosylation of protein targets with the identification of ART enzymes involved in antibiotic inactivation by bacteria and DNA damage repair in eukaryotes. In this talk, I will present our discovery and characterization of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. An X-ray crystal structure of RhsP2 reveals that it resembles protein-targeting ARTs such as Diphtheria toxin. Remarkably, however, RhsP2 does not act on a protein target and instead ADP-ribosylates the unique 2′-hydroxyl group of RNA. Besides exhibiting a preference for double-stranded RNA, we find that the RhsP2 activity is highly promiscuous with identified cellular targets including numerous non-coding RNAs, including the entire tRNA pool and the essential RNA-processing ribozyme, RNase P. Further characterization of RhsP2-intoxicated cells indicates that target cell death arises from the simultaneous inhibition of protein translation and disruption of RNA processing. Overall, our data demonstrate a new mechanism of bacterial antagonism and uncover an unprecedented enzymatic activity catalyzed by the ART family of enzymes.
Sunday
Intermission
09:45am - 10:00am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

Sunday
3736507 - Spatial quantification of cyclic dimeric guanosine monophosphate and related biosynthetic products in Vibrio cholerae biofilm colonies
10:00am - 10:45am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Vibrio cholerae causes the diarrheal disease cholera, affecting millions of people worldwide every year. V. cholerae adapts to environmental stressors as it passes through the environment and enters a host by producing biofilms. Although biofilms are connected to virulence, the exact mechanism by which dispersal from a biofilm state leads to increased virulence is not known. In V. cholerae, the second messenger molecule, cyclic dimeric guanosine monophosphate (c-di-GMP), controls motility and biofilm production, and represses virulence. Current methods for detecting c-di-GMP include fluorescent assays and LC-MS analysis as bulk measurements that do not allow for correlating presence of c-di-GMP with biofilm structures. Additionally, neither method allows for the simultaneous detection of additional chemical signals which may be influenced by c-di-GMP. V. cholerae possesses a large number of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) for synthesizing and degrading c-di-GMP in response to environmental changes such as temperature and oxygen levels. Here we use MALDI-qTOF imaging mass spectrometry (IMS) to detect both relative and quantitative c-di-GMP levels spatially across V. cholerae biofilm colonies. Our approach allows for the detection of spatial variation in c-di-GMP levels in agar colony biofilms, which is currently only possible using fluorescent detection as an indirect measurement that relies on a reporter system. Absolute quantification of c-di-GMP is possible by LC-MS, however the spatial heterogeneity of c-di-GMP production is lost. We have optimized MALDI matrix selection, sample preparation, and MS parameters to enhance the detection of c-di-GMP while maintaining untargeted m/z signals for other small molecules. As a second messenger, c-di-GMP influences many different cellular processes and with this approach we aim to contextualize c-di-GMP production by associating it spatially with co-expressed chemical signals. We focus on c-di-GMP metabolism as the central signaling system for biofilm production, and identify chemical signals that are spatially associated with c-di-GMP metabolism and biofilm secretion. This approach builds a foundation for studying the complex chemical signaling cascades that may affect V. cholerae in a host environment.
Sunday
3738298 - Selective probes for targeting bacterial Hsp70 chaperones to potentiate antibiotics
10:45am - 11:30am USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Tania Lupoli, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Bacterial DnaK belongs to the ATP-dependent heat shock protein 70 (Hsp70) family of chaperones, which catalyze nascent protein folding and salvage misfolded proteins. DnaK collaborates with cofactors, DnaJ and GrpE, which regulate its catalytic cycle and conformational state. In the bacterial pathogen Mycobacterium tuberculosis, DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein-protein interactions between DnaK and at least one DnaJ is essential for survival of mycobacteria; hence, chaperone-cofactor interactions may represent untapped antibacterial targets. Some Hsp70 inhibitors show cross-reactivity with bacterial homologs, but we lack selective probes for bacterial DnaK. Here, we describe unbiased and rational approaches to modulate bacterial DnaK ATPase and chaperone activity, with the goal of designing probes with improved specificity that may be used to assess DnaK as an antibiotic target. First, we detail the discovery of small molecule inhibitors of mycobacterial DnaK from a high through-put screen, most notably a peptidomimetic called telaprevir, which is able to inhibit chaperone function through interactions with the peptide-binding cleft of DnaK. Binding of telaprevir leads to conformational changes that prevent ATP hydrolysis in a distal domain. Using in vitro and in vivo chaperone assays, we demonstrate that telaprevir allosterically modulates the function of mycobacterial DnaK and its cofactor protein DnaJ2. As part of an orthogonal approach, we synthesize protein fragments that mimic a crucial N-terminal domain of mycobacterial DnaJ proteins in order to disrupt DnaK-DnaJ interactions. We find that proteomimetics of DnaJ1 are able to inhibit DnaK activation by cofactors in vitro, and cause cell death when mycobacterial cells are exposed to heat, a common proteotoxic stress. Similarly, while telaprevir alone is not toxic to mycobacteria, co-treatment of cells with telaprevir and aminoglycosides, which further stress the proteome, enhances the potency of these antibiotics. In addition, telaprevir combats mycobacterial resistance to the frontline TB drug rifampin, as DnaK-DnaJ2 function is required for stabilization of protein mutants that confer drug tolerance. Collectively, our work sets the stage for the design of peptide-based inhibitors with improved selectivity for bacterial chaperones to probe chaperone-protein interactions and explore chaperone inhibitors as adjuvants for different classes of antibiotics.
Emerging Areas & New Methods in Biological Chemistry:
08:00am - 11:55am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Phoebe Glazer, Organizer; Ekaterina Pletneva, Organizer, Presider; Amanda Hargrove, Presider
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
3754940 - New HD LC MS methods of characterizing concentrations H2S and its oxidized metabolites in cell cultures.
08:00am - 08:20am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Hydrogen sulfide (H2S) is an endogenous signaling molecule which plays important roles in physiological functions of the cardiovascular system, but it’s mechanism of action is poorly understood. We have translated a common methodology to assess protein sulfenic acids to characterize and quantify endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in numerous cell lines and mammalian tissue samples. The method using nucleophilic and electrophilic traps for H-S and HO-S bonds to trap small molecular weight species, and allows quantification of these species giving both intra- and extra-cellular concentrations based on an H2S calibration curve. We compare the production of H2S and SOS across human, bacteria and yeast cell lines, as well in primary human human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. H2S and its metabolites are orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar) and yeast (femtomolar). We proposed the sulfomic index as a profile of non-protein S-oxide redox status. H2S and SOS were also found to be effluxed from smooth muscle cells in higher concentrations than endothelial cells in similar ratios. Human aortic smooth muscle cells were selected to examine changes in intracellular and efflux concentrations of H2S and SOS under hypoxic growth conditions. Under hypoxia, the smooth muscle cells demonstrated reduced mitochondrial respiration and increased ROS production, with a corresponding reduction in levels of H2S and HOSOH, but large increases in levels of HSOH, which we attribute to retrograde electron transfer to Complex 1 during hypoxia. We believe that measurements and characterizations these analytes under different disease states may help uncover the mechanisms of action attributed to H2S in cardiovascular health and disease. Several collaborators provided assistance and materials in this work and will be acknowledged in the presentation.
Sunday
3739446 - Profiling sulfur(VI) fluorides as reactive functionalities that expand the ligandable proteome
08:20am - 08:40am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Katharine Gilbert, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Expanding the range of biological systems that can be investigated and targeted by chemical biology approaches is becoming increasingly crucial to the development of novel therapeutics. Reactive electrophiles capture specific compound-target interactions, so can be leveraged as tools to facilitate the discovery and development of new medicines. Current reactive strategies suffer from limited applicability across the proteome, so expanding the range of biological systems that are amenable to targeting remains a key challenge. Sulfur(VI) fluoride (SVI-F) reactive electrophiles target numerous protein residues, so can be leveraged for the covalent modification of a broad scope of protein targets. SVI-F functionalities can exhibit diverse reactivity that can compromise the desired modification, yet to date, SVI-F functionalities have been incorporated into ligands with minimal rational design. Here, we present a three-step workflow to profile SVI-F functionalities (Figure 1) to enable the knowledge-guided application of SVI-Fs with optimal reactivity.

Our SVI-F profiling workflow has identified an ideal reactivity space for SVI-F electrophiles and further enabled the reactivity prediction of novel SVI-F electrophiles. This work has examined the interplay between the reversible engagement and irreversible modification steps, and demonstrated the efficacy of SVI-F tools in both recombinant proteins and live cells, and capacity to modify an expanded range of protein targets compared to current strategies. Our SVI-F profiling workflow is envisioned to expand the utility of SVI-F functionalities in chemical biology.

Sunday
3755104 - Organelle-targeted iron ligands to elucidate cellular metal metabolism and ferroptosis
08:40am - 09:00am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Coordination chemistry of biometals in the cell is readily studied with the help of appropriate chelators and indicators. Disease processes often involve mislocation and overload of iron, which can lead to reactive oxygen species production that damages biomolecules. We have focused on novel chelators for iron bioconjugated to organelle-targeting peptides in order to further biochemical understanding of disease pathways. New designs and syntheses of tripodal ligands to sequester iron and affect the formation of hydroxyl radical in the cell are presented. For mitochondrial targeting, several amphipathic peptides related to the Szeto-Schiller family are being studied. We report success in a peptide design that delivers an Fe(II)/Fe(III) ligand and a fluorophore to the inner membrane. The endoplasmic reticulum is also coming under study. This chemistry has a connection to ferroptosis, a relatively new programmed cell death process.
Sunday
3745736 - Fluorogenic bifacial PNAs (bPNAs) as a new probe for intracellular tracking of RNAs
09:00am - 09:20am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Dennis Bong, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

New methods are needed for RNA tracking that are convenient, compatible with live cell tracking and minimally perturbative to RNA structure and biology. We demonstrate herein that fluorogenic bPNAs are useful tools for intracellular labeling of RNAs, RNA-protein complexes and live cell tracking of genomic loci. In particular, bPNAs N-terminally capped with fluorogenic dye exhibit significant (~500X) enhancement of emission intensity upon bPNA triplex hybridization with structured RNAs bearing U-rich internal bulges (URILs) in vitro; further, bPNAs are known to be efficiently taken up into mammalian cells in culture. Stem replacement labeling in folded RNAs was carried out by installation of U4xU4 URILs and the constructs introduced into mammalian cell culture by plasmid transfection. Treatment in culture with fluorogenic (Fl) bPNAs yielded in 5-10X enhancement green fluorescence emission in cells expressing the URIL RNA over cells without URIL expression. Using known RNA binding protein partners and RNA sequences, we found that Fl-bPNA labeling of URIL-RNA corroborated by RBPs fused to red fluorescent proteins (FP). Co-localization of red (FP) and green (Fl-bPNA-RNA hybrid) fluorescence was only observed with the correct pairing of RNA and RBP. We demonstrated the application of this strategy to genomic loci tracking by the URIL modification of sgRNA in dCas9 Crispr complexes in live U2-OS cells. All intracellular RNA labeling was benchmarked against MS2/MCP labeling, which confirmed bPNA tracking. Together, these experiments show that fluorogen-tagged bPNAs are useful for intracellular stem-replacement labeling of RNAs as well as tracking of RNA and RNA-protein interactions. Given the relatively modest perturbation to RNA structure afforded by stem replacement of a native 4 bp stem with a URIL, we anticipate that Fl-bPNA may find a broad scope of applications in RNA tracking.

Sunday
3738270 - Ligand tuning for 64cu Positron Emission Tomography (PET) imaging in alzheimer's disease
09:20am - 09:40am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Positron Emission Tomography (PET) imaging agents for Alzheimer’s Disease currently utilize 11C or 18F radionuclides, the incorporation methods and short half-lives of which make difficult their widespread clinical use. We have sought to create ligands suitable for complexation with Cu-64 to serve as alternative PET imaging agents for AD with longer half-lives and easier methods of radionuclide incorporation. Modification of a triazacyclonone (TACN) backbone by variations in the N-linked fragments allows for ‘tuning’ of several ligand properties. Ligand tuning via two mechanisms has been explored: the number of amyloid-targeting fragments and the size of aliphatic substituents. By varying the number of amyloid-targeting fragments, the beneficial effect of incorporating a second amyloid-targeting fragment to increase affinity for amyloid plaques is weighed against the drawback of ligand size. By varying the size of the aliphatic substituent, the benefit of increased aliphatic nature of the ligand can be measured and weighed against the drawback of increased bulk close to the Cu-chelating pocket of the ligand. Physical and in vivo characterization of the derivatized ligands has allowed for determination of the PET imaging potential of the ligands in relation to the ‘tuning’ of their physical structure
Sunday
Intermission
09:40am - 09:55am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Sunday
3742443 - Organelle temperature measurements using anisotropy-based nanothermometers
09:55am - 10:15am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Temperature is a crucial parameter in biology, medicine, and physics. Because of that, in the last years, several methods have been developed and presented to measure nanoscale temperature. Optical methods excel because they are non-invasive, spatially accurate, and can measure real-time local changes in temperature. Among these, fluorescence anisotropy-based methods are particularly advantageous because they are less affected by changes in the probe concentration and irradiation conditions. I will present intracellular temperature measurements in cancer cells and live organisms using the green fluorescent protein and the theoretical and practical method to add thermosensitivity to any protein and DNA. In addition, it will also discuss the measurements of the organelle-oriented intracellular temperature of cancer cells for cancer diagnostic and treatment. I will focus on two of the most important cell organelles: Nucleus and lysosomes. Using these nanothermometers by a broad spectrum of disciplines within the scientific community will bring new intracellular knowledge and understanding that today remains unavailable with current techniques.

Sunday
3743106 - Chemical tools to control dopamine and serotonin receptors: focus on intracellular signaling and proteomics-based target ID
10:15am - 10:35am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Dopamine and serotonin receptors are central to brain function, yet are extremely challenging to study, especially in precise regions and narrow timescales. This is because traditional drugs for these receptors are subject to diffusion and other pharmacokinetic and pharmacodynamic factors that limit the spatiotemporal precision of experimental outcomes. While there are several excellent methods to control neurons, they all rely on genetic manipulation, and methods to create orthogonally-controlled non-mutant receptors are lacking. Our ultimate goal is to create new tools to study dopamine and serotonin GPCRs in living mammals, without the need for genetic manipulation. Here, we report on the synthesis of new probe molecules for dopamine receptor D2, and the probes' biochemical and proteomic analysis. We also report on new probes to control the serotonin receptor HT2C.
Sunday
3738067 - Bifunctional molecules as prodrugs for the immunoproteasome OnDemand
10:35am - 10:55am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Targeting a diseased cell population over a healthy cell population in drug discovery still remains a challenge. Prodrugs have been effective in targeted drug release to improve a parent drug’s off-target effects and pharmacokinetic profile. We are interested in developing hybrid molecules that contain an unnatural peptide sequence linked to a biologically active molecule. The peptide sequence has been designed to interact with the immunoproteasome (iCP). The iCP is an isoform of the proteasome that is overly expressed when a cell is infected or introduced to inflammatory cytokines, such as interferon-gamma and tumor necrosis factor-alpha. Once the hybrid molecule binds to the chymotrypsin-like active site of the iCP, the biologically-relevant molecule will be released from the peptide. Our initial results indicate this approach can release a number of cytotoxic molecules mainly in the cells expressing the iCP. The sequence of the peptide can also be modified depending on the desire to also inhibit excessive iCP function. Further medicinal chemistry optimization and biochemical characterization has further allowed us to better understand what functional groups are favored by the iCP’s substrate channel for more efficient cleavage and therefore, activation of our prodrug-like scaffold.
Sunday
3741802 - Complex metal oxide nanomaterials directly generate as well as induce reactive oxygen species production in bacteria upon chronic exposure
10:55am - 11:15am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Complex metal oxide nanoparticles are of immense use as the demand of more sustainable solutions emerge like electric vehicles. These nanomaterials during their manufacture, use and disposal eventually make their way to the environment and have interactions with various organisms including single cellular to multicellular organisms. Focusing on an environmentally relevant bacterial species Shewanella oneidensis MR-1 found ubiquitously in nature and mainly responsible for bioremediation of heavy metals, we have assessed the toxic effects of Lithiated Nickel Manganese Cobalt oxide (NMC) which is an emerging battery cathode material for electronic devices and electric vehicles. We have reported earlier that brief exposure of NMCs to S. oneidensisis lethal for the bacteria but chronic exposure continuously for multiple generation results in the emergence of adaptive phenotype where bacteria is able to tolerate the otherwise lethal concentration of NMC. In the present study, we delineate the variations during the adaptation process in terms of reactive oxygen species (ROS) levels and changes in phenotype of the NMC adapted bacterial population. These adapted population also show the presence of membrane vesicles containing ROS as well as possess the propensity of generation random mutations in DNA as well as DNA damage. Our study reflects on the process of adaptation and resistance generation in bacterial species due to the presence of toxic complex metal oxide nanomaterials.

Sunday
3747188 - Development of hybrid compounds of peptides with metal complex-type and triptycene-type core units that induce paraptosis in cancer cells
11:15am - 11:35am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Programmed cell death (PCD) plays an important role in the formation of tissues and organs, and literally cell death (apoptosis, necroptosis, paraptosis, autophagy, etc.). To date, many anticancer drugs have been reported to induce PCD in cancer cells. In order to overcome the resistance of cancer cells to anticancer drugs, it is necessary to develop different PCD-inducing drugs and to study their mechanisms.
In this work, we carried out the design and synthesis of the hybrid compounds of Ir(III) complexes with amphiphilic basic (positively charged at neutral pH) peptide (AP-IPHs, AP = amphiphilic peptide, IPH = Ir complex-peptide hybrid), which function as inducers of paraptotic cell death in cancer cells and detectors of dead cancer cells. Mechanistic studies of cell death induced indicate that AP-IPHs induce the direct transfer of Ca2+ from the endoplasmic reticulum (ER) to mitochondria, the loss of mitochondria membrane potential, membrane fusion between the ER and mitochondria, and the vacuolization of intracellular organelles in cancer cells. Paraptosis induced by IPHs has been referred to as paraptosis-II, which is different from previously known paraptosis (paraptosis-I) induced by celastrol and other natural products, in which the Ca2+ overload in mitochondria is negligibly induced.
Very recently, triptycene units (not luminescent) were developed very recently for the assembly of three KKKGG peptide units instead of Ir(III) complex cores and it has been found that these triptycene-peptide hybrids (TPHs) induce PCD in Jurkat cells and other cancer cell lines via similar intracellular signaling pathways. These results will be reported in this presentation.

Sunday
3742159 - Withdrawn
11:35am - 11:55am USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Postdoctoral Fellow Symposium:
02:00pm - 05:30pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Phoebe Glazer, Organizer; Ekaterina Pletneva, Organizer; Amanda Hargrove, Presider
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
3734564 - Non-lamellar lipid structures as artificial organelles
02:00pm - 02:15pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Cells regulate their biochemical processes using structured lipid assemblies as compartments. Membrane-bound organelles concentrate and colocalize molecules to favor specific biochemical processes while isolating incompatible reactions. Many studies have attempted to create experimental model systems to emulate subcellular organelles organization using lamellar lipid vesicles. However, organelles are often arranged into highly convoluted morphologies in which lipid bilayers form non-lamellar periodic structures, providing large reaction spaces for biological transformations. Despite the importance of non-lamellar lipid architectures in cells, in-depth investigations of organelle functions are hampered by the scarcity of methods for preparing and programming synthetic non-lamellar membranes.
Here, we present artificial lipid compartments based on a zwitterionic gemini surfactant that spontaneously self-assembles into non-lamellar lipid sponge phase droplets in aqueous media. We have developed a robust bioconjugation methodology to program non-lamellar lipid assemblies to recruit and concentrate biologically relevant molecules such as transmembrane proteins and soluble enzymes.
We incorporated benzylguanine (BG)-modified phospholipids into lipid sponge phase structures capable of forming stable covalent bonds with O6-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins, enabling programmable control over protein capture. As a model protein for the SNAP bioconjugation, we chose a SNAP-tag superfolded green fluorescent protein (SNAP-sfGFP), which was incubated with lipid sponge phase droplets bearing 1 mol% of BG-modified phospholipids for 15 min at room temperature. Protein capture was confirmed by fluorescence confocal microscopy and high-resolution mass spectrometry analyses. The structure of the lipid droplets was studied by small-angle X-rays scattering (SAXS) analysis, showing the presence of a non-lamellar sponge bi-continuous phase (L3). We have demonstrated that SNAP-tag technology could be used to direct protein sequestration in the presence of complex transcription-translation biochemical machinery required for cell-free protein expression.
Our results show that lipid sponge phase droplets can be programmed to integrate proteins that regulate complex biological functions and should facilitate the development of advanced biomimetic artificial organelles.

Sunday
3753138 - Structural insights into bacterial sterol transporters OnDemand
02:15pm - 02:30pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Sterol lipids, like cholesterol, are widely present in eukaryotes and play critical roles in a variety of physiological processes. These include signaling and maintaining the structural integrity of the cell membrane. Aberrant homeostasis leads to many human diseases. Studies on sterols lipids were almost exclusive to eukaryotes, but a growing body of work suggests that some bacteria species are also capable of producing sterols. One example is the aerobic methanotroph Methylococcus capsulatus, which is known to synthesize 4-methylsterols from 4,4-dimethylsterols; the de-methylated lipid resides in its outer membrane. It is crucial to traffic the methylsterol products to the outer membrane to maintain homeostasis in the cytoplasm. Despite this discovery four decades ago, the lack of knowledge about bacterial sterol transporters obscured the trafficking of 4-methylsterol. In this work, we used a combination of bioinformatics, biophysical and structural techniques to identify three novel sterol transporters in Methylococcus capsulatus that can bind to 4-methysterols. Our study provides the first crystal structures of innate sterol transporters in the bacterial domain, reveals their structural bases for sterol binding, and describes their pronounced structural divergence from eukaryotic sterol transporters. Together, the work expands our understanding of the functional and evolutionary roles of these lipids in bacteria.
Sunday
3735750 - Discovery of a degron for the thalidomide-binding domain of cereblon through a targeted protein degradation strategy
02:30pm - 02:45pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Targeted protein degradation induced by small-molecule degraders has emerged as an attractive therapeutic approach to eliminate disease-relevant proteins from cells. Among the >600 E3 ligases encoded by the human genome, cereblon (CRBN), a substrate recognition adaptor in the CRL4CRBN E3 ubiquitin ligase complex, plays a central role in targeted protein degradation applications. CRBN is a target of thalidomide and lenalidomide, which are clinically used in the treatment of hematopoietic malignancies and as ligands for targeted protein degradation. Despite expanding development of CRBN-targeting therapeutics, the endogenous structural motif recognized by the thalidomide-binding domain of CRBN has remained elusive. E3 ubiquitin ligase complexes select proteins for degradation through the recognition of degrons, specific amino acid sequences that are sufficient to promote ubiquitylation and degradation when embedded in a substrate. We thus hypothesized that synthetic ligands of CRBN, thalidomide and lenalidomide, mimic a naturally occurring degron of CRBN. Here, we will discuss our approach to discovering a degron for the thalidomide-binding domain of CRBN using a targeted protein degradation strategy as a functional readout.
Sunday
3741228 - Novel expression system for the expression, purification, and characterization of recombinant human heteropolymer ferritin of different H to L subunit ratios
02:45pm - 03:00pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Mammalian ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types named H and L. These subunits co-assemble in various H to L ratios to form shell-like protein structures (isoferritins) of ~ 8 nm inner cavity diameter able of accommodating thousands of iron atoms in the form of biologically available hydrous ferric oxide mineral core. Despite their discovery more than 8 decades ago, recombinant isoferritin nanostructures have never been synthesized, owing to a lack of a good expression system. Earlier in-vitro reconstitution attempts using chemical denaturation and unfolding of recombinant homopolymers H- and L-subunits followed by their renaturation have either largely failed or yielded very low amounts of heteropolymer ferritins that are not representative of those occurring naturally. Here, we describe for the first time a unique approach that uses a novel plasmid design that enables the synthesis of these complex ferritin nanostructures. Our study reveals an original system that can be easily tuned by altering the concentrations of two inducers, allowing the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between. The H to L subunit composition of purified ferritin heteropolymers was analyzed by SDS-PAGE and capillary gel electrophoresis, and their iron handling properties were characterized by light absorption spectroscopy. Our results provide important insights into heteropolymer ferritin structure-function relationships and the role of each subunit in the proper management of iron
Sunday
3741332 - Structure of lactate oxidase from Enterococcus hirae revealed a new aspect of active site loop function: substrate/product inhibition
03:00pm - 03:15pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
L-lactate oxidase (LOx) is a flavin mononucleotide (FMN) dependent TIM barrel fold enzyme that catalyses a reductive half-reaction in which L-lactate is oxidized to pyruvate by FMN reduction as well as an oxidative half-reaction in which the reduced FMN is reoxidized by molecular oxygen coupled with H2O2 production. For its high activity, stability, and specificity, LOx has been utilized to industrial applications. The substrate and product inhibitions has been recognized as one of the characteristic features of LOx, however its mechanism has yet to be elucidated.
In this study, X-ray structure of LOx from Enterococcus hirae (EhLOx) was determined at 1.70 Å (PDB ID; 6M73) resolution. EhLOx was produced by E. coli recombinant production system. The purified EhLOx was co-crystallized with 0.4 M sodium pyruvate and X-ray diffraction data were collected at the Photon Factory BL-5A. Surprisingly, the resulting structure revealed a complex of EhLOx with a D-lactate form ligand covalently bonded with Tyr211, which located on loop 4 (Figure). It suggests that a deprotonated Tyr211 would attack on α-carbon of pyruvate and form covalently bound D-lactate form ligand, whereas D-lactate is known as an inhibitor of LOx. The elucidated complex structure might be caused by high concentration of pyruvate, consequently the results may represent the substrate/product-inhibition mode of LOx with D-lactate. These findings gave us new suggestions that the loop 4 is involving in substrate/product inhibition mechanism of LOx.
Figure. EhLOx structure and its active site.

Figure. EhLOx structure and its active site.


Sunday
3755301 - An orthogonal replication system for continuous evolution of proteins in E. coli
03:15pm - 03:30pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
The development of an orthogonal DNA replication system comprised of a ‘replisome plasmid’ (RP, maintained by the host) and an orthogonal ‘episome plasmid’ (EP, maintained by the RP, not the host) in E. coli allows for major repurposing of the exogenous replisome’s mutation rate beyond the host’s genomic error threshold for continuous evolution of genes of interest. Orthogonal replication in E. coli has been described for several lytic coliphages that encode their own replisome. However, such phages kill the host upon infection and thus do not require mechanisms to maintain their copy number at a level sustainable for the host organism. In contrast, lysogenic phages have evolved strategies to control the copy number of their genomes after infection to achieve prolonged lysogeny with their host, however these phages rely on the host replication machinery. My strategy for developing an orthogonal replication system in E. coli is therefore based upon an RP based on the orthogonal replisome of a lytic phage (bacteriophage T7) and an EP based on the control mechanisms of a lysogenic phage (bacteriophage N15).
T7 replication involves five essential proteins: T7 DNA polymerase, DNA helicase/primase, the ssDNA binding protein, T7 RNA polymerase, and the host factor E. coli thioredoxin. I have designed an RP comprising the T7 replisome. To confirm that all genes are functional, E. coli. strains that had been transformed with the RP have been infected with T7 phages in which the four essential phage replication genes had been knocked out. In all cases, phage growth was recovered, demonstrating that the expressed replication proteins are functional.
The T7 replisome is highly processive and replication of circular plasmid DNA leads to formation of toxic concatemeric DNA through rolling circle replication. To avoid this, I chose a linear EP based on a lysogenic N15 phage vector. The N15 protelomerase TelN cuts DNA at specific recognition sequences at both ends of the linear plasmid to form covalently closed ends at the cleavage site, thus preventing formation of high molecular weight DNA by rolling circle replication I have cloned the EP with the T7 origin of replication, as well as the N15 protelomerase. Maintenance of the EP by the RP has been confirmed. Mutations of T7 DNA polymerase mutants lacking its proofreading exonuclease activity, as well as additional mutations in the palm domain are investigated for increasing the mutation rate of the orthogonal replisome.
]

Sunday
3738802 - Withdrawn
03:30pm - 03:45pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Session Type: Oral - In-person

Sunday
3754601 - Cell-free prototyping and rapid optimization of paper-based biological sensors
03:45pm - 04:00pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Cell-free systems enable the use of biological mechanisms without the need to maintain cell efficacy and thus enable the creation of low burden and fieldable detection platforms for nucleic acids, small molecules, and toxins. However, designing and optimizing portable cell-free biological sensors for clinical and environmental applications is challenging due to the complexity of the system and lack of high-throughput workflows. Here we describe a method to accelerate the in vitro prototyping and optimization of paper-based cell-free sensors using high-throughput acoustic liquid handling in a 384-well format. This methodology allows for lyophilized cell-free tickets with varying formulations of energy sources, cryoprotectants, and cofactors to be prepared and measured in mass to assess biosensor performance. We show how this system coupled to machine learning accelerates design–build–test cycles and improves performance in β-galactosidase based biosensors.
Sunday
Intermission
04:00pm - 04:15pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

Sunday
3734579 - Antibacterial activity of metergoline analogues and investigations into their mechanisms of action
04:15pm - 04:30pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Metergoline is a semisynthetic ergot alkaloid identified recently as an inhibitor of Salmonella Typhimurium, an intracellular Gram-negative pathogen. A series of carbamate, urea, sulfonamide, amine, and amide analogues of metergoline were synthesized and evaluated against Gram-positive and Gram-negative bacteria. Cinnamide and arylacrylamide analogues showed improved potency relative to metergoline, and one analogue was effective against MRSA in a murine skin infection model. Arylacrylamide derivatives were inactive against wild-type Gram-negative bacteria but their potencies were improved considerably (up to >128-fold) in combination with outer-membrane permeabilizer SPR741. Metergoline analogues also retained activity against multidrug-resistant strains of Staphylococcus aureus, suggesting a mechanism of action that is distinct from clinically used antibiotic classes.

Sunday
3744238 - Chiral cerium oxide nanoparticles for antibacterial activity
04:30pm - 04:45pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Nanoparticles (NPs) have been considered as antimicrobials for a long time; the success of clinical studies of these NPs primarily relies on the interaction ability with the bacteria. Therefore material, shape, size, charge, and surface chemistry are important. These features give them a selective antibacterial activity that is needed for next-generation antibiotics. In this study, we used chiral ceria NPs for antibacterial activity on Staphylococcus aureus and Escherichia coli for selective inhibition. Even though most of the studies on antimicrobial NPs suggest reactive oxygen species (ROS) formation and ion release as the primary source of antibacterial activity, the mechanism of NP’s antibacterial action is remarkably more complex than generating ROS or the release of ions. This complexity originated from the biomimetic function of these NPs that can be designed to make complex with bacterial membrane proteins using unified structural descriptors. Yet, further investigation into the antimicrobial mechanisms of these biomimetic NPs is essential for future clinical translation.
Sunday
3730958 - Withdrawn
04:45pm - 05:00pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

Sunday
3753813 - Size and shape controllable synthesis of seedless gold nanoparticles for the development of immunochromatographic assay
05:00pm - 05:15pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Gold nanoparticles (AuNPs) of different shapes and sizes represent significant color changes in a reaction mixture that act as a promising biosensing tool to develop the rapid Lateral Flow Immunochromatographic Assay (LFIA). Herein we report that the growth transformations of AuNPs are a function of physicochemical reaction parameters. Surface Plasmon Resonance of anisotropic nanoparticles is dependent on their morphological features and geometry. In this study, we have synthesized the seedless AuNPs by using various molar ratios of HEPES and HAuCl4. The shape and the geometry of the gold nanostructures were modulated under the influence of pH (5, 7, and 9) of the HEPES, molar concentrations of HEPES to HAuCl4, and temperature ranges (20 °C, 40 °C, and 60 °C). The change in the color of reaction mixtures over time was recorded in terms of the absorbance of the UV-Visible light in the range of 300-800 nm of the light wavelength. The scanning transmission electron microscopic images employed that the gold nanostructures exist in various shapes and corroborate with the UV-Vis spectrum. It was confirmed that the nano-moieties are anisotropic, stable, and exist in the size range of 1 nm to 100 nm. The present study confirms that the change in the color of nanostructures reactions mixtures is a function of the surface plasmon resonance bands under the influence of physicochemical reaction conditions. Physicochemical parameters such as temperature, pH, the molar concentration of the reactants act synergistically to influence the reaction kinetics, molecular mechanics, and enzymatic catalysis that aid to affect the size, shape, and biochemical corona of nanoparticles. The findings of the present study explain the growth kinetics of the AuNPs of various colors that can be observed by unaided eyes and have promising applications for the development of the rapid LFIA for the detection of mycotoxins in food samples.
Change in the color of the reaction mixture with the increasing concentration of disodium phosphate.

Change in the color of the reaction mixture with the increasing concentration of disodium phosphate.


Sunday
3742780 - Inhibition of measles viral fusion is enhanced by targeting multiple domains of the fusion protein
05:15pm - 05:30pm USA / Canada - Central - August 21, 2022 | Location: Glessner House C (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Measles virus (MeV) infection remains a significant public health threat despite ongoing global efforts to
increase vaccine coverage. As eradication of MeV stalls, and vulnerable populations expand, effective antivirals against MeV are in high demand. Here, we describe the development of an antiviral peptide that targets the MeV fusion (F) protein. This antiviral peptide construct is composed of a carbobenzoxy-DPhe- L-Phe-Gly (fusion inhibitor peptide; FIP) conjugated to a lipidated MeV F C-terminal heptad repeat (HRC) domain derivative. Initial in vitro testing showed high antiviral potency and specific targeting of MeV F-associated cell plasma membranes, with minimal cytotoxicity. The FIP and HRC derived peptide conjugates showed synergistic antiviral activities
when administered individually. However, their chemical conjugation resulted in markedly increased antiviral potency. In vitro mechanistic experiments revealed that the FIP−HRC lipid conjugate exerted its antiviral activity predominantly through stabilization of the prefusion F, while HRC-derived peptides alone act predominantly on the F protein after its activation. Coupled with in vivo experiments showing effective prevention of MeV infection in cotton rats, FIP−HRC lipid conjugates show promise as potential MeV antivirals via specific targeting and stabilization of the prefusion MeV F structure.

Abeles & Jencks Award:
02:00pm - 06:00pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Phoebe Glazer, Organizer; Ekaterina Pletneva, Organizer, Presider
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
Introductory Remarks
02:00pm - 02:05pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Sunday
3751774 - Structure-guided insights into specificity, mechanism and evolution in bacterial glycoconjugate biosynthesis
02:05pm - 03:00pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Karen Allen, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Bacterial glycoconjugates are structurally diverse macromolecules that mediate critical interactions among bacteria and between bacterial pathogens and their hosts. Many glycoconjugates are biosynthesized using a common biosynthetic strategy involving en bloc transfer of glycan to proteins, lipids, or other glycans. These membrane-committed pathways are initiated by a polyprenol phosphate-phosphoglycosyl transferase (PGT). The following steps are mediated by glycosyl transferases (GTs), acting on membrane-resident polyprenol diphosphate-derivatives (Figure 1). The X-ray crystal structure of the PGT from Campylobacter concisus, PglC (2.74 Å resolution), shows that the monotopic PGTs include a reentrant membrane helix that penetrates one leaflet, then re-emerges. Molecular dynamics simulations show the undecaprenol phosphate (UndP) carrier also occupies a single leaflet and allows a first view of UndP binding to PglC, associated with induced fit. Bioinformatic analysis of the monotopic PGT superfamily uncovered extensive fusions with other pathway enzymes and provides evidence that these enzymes are structured to gather rare substrates. The structure of the enzyme that carries out the next step, addition of N-acetyl-D-galactosamine, PglA, (determined at 1.9 Å resolution) shows remarkable similarity to PglH (rmsd 1.9 Å), which catalyzes the processive addition of three N-acetyl-D-galactosamine moieties in the penultimate step. Differences between the GTs PglA and PglH with respect to the relative orientations of the active site to the membrane interface suggest that membrane positioning may play an integral part in specificity.
<b>Figure 1.</b> Biosynthesis of a polyprenol disphosphate-linked glycans by PGT and GT enzymes via the membrane-associated pathway.

Figure 1. Biosynthesis of a polyprenol disphosphate-linked glycans by PGT and GT enzymes via the membrane-associated pathway.


Sunday
3754330 - Basis and origin for asymmetric trimers in the tautomerase superfamily: Analysis and implications
03:00pm - 03:35pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Christian Whitman, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Tautomerase superfamily (TSF) members repurpose a simple β-α-β unit to make a collection of structures (homo-, heterohexamers, and trimers) that carry out an assortment of activities (primarily tautomerization, dehalogenation, hydration, and decarboxylation reactions). Recently, an asymmetric trimer was discovered where one monomer is flipped 180° relative to the other two monomers to create three different active sites. Since these trimers might provide an additional scaffold for diversification (along with the symmetric trimer), the basis and origins for the asymmetry were investigated. The results show (1) the number of salt bridges governs whether a trimer is symmetric (in A) or asymmetric (in B) and (2) suggest that fusion of an asymmetric heterohexamer led to an asymmetric trimer.

Sunday
Intermission
03:35pm - 04:15pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Sunday
3754135 - Structural features of the Ras/Raf interface in the context of Ras dimerization
04:15pm - 04:50pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Carla Mattos, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Ras is a master signaling GTPase that is found mutated in nearly 20% of human cancers due to its role in controlling cell processes usurped during cancer evolution, including the MAPK proliferative pathway. It has been a challenging drug target in part due to limited understanding of the molecular mechanisms through which it propagates signaling. Thus, it is critical that we focus on understanding the fundamental features of how the wild type protein works. The Raf Ras binding interface consists of two domains: the high affinity Ras Binding Domain (RBD) and a low-affinity cysteine rich domain (CRD). We and colleagues have recently shown that the RDB promotes robust dimerization of Ras on supported lipid bilayers. A dimer with Ras a4-a5 interface appears in a large number of Ras crystal structures as well as in structures of Ras/Raf-RBD and Ras/Raf-RBD-CRD. We use these structures in conjunction with MD simulations to develop a novel model of Ras signaling through Raf. In this model, the two RBD molecules across the dimer interface are linked through strong allosteric connections modulated by the CRD, possibly to promote binding of the scaffold protein galectin, which itself is a dimer. Our model envisions a polymeric signaling platform composed of Ras/Raf and galectin dimers in which GTP hydrolysis does not involve GAP proteins. Instead, the dimer stabilizes a calcium binding allosteric site that modulates GTP hydrolysis on the Raf-bound Ras dimer. Signaling and GTP hydrolysis are decoupled in this model with signaling turned on by Ras-GTP binding Raf, forming the dimer and recruiting galectin; and turned off by enhanced GTP hydrolysis modulated by calcium binding. This model provides a new paradigm for understanding and targeting signaling through the MAPK pathway.
Sunday
3754181 - Searching for mechanistic differences in the hydroxylating and desaturating L-Arg oxidases
04:50pm - 05:25pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
MppP from Streptomyces wadayamensis (SwMppP) is a pyridoxal-5’-phosphate (PLP)- and oxygen-dependent enzyme that oxidizes L-arginine to 4S-hydroxy-2-ketoarginine in the biosynthesis of the non-proteinogenic amino acid L-enduracididine (L-End). L-End is a key building block of several nonribosomally produced peptide antibiotics, such as the cyclic glycopeptide mannopeptimycin. Our previous work established a plausible mechanism for this unusual reaction, and that MppP is an oxidase rather than an oxygenase. We then began to look at other MppP homologs to see in what other biochemical contexts this activity appears. A PHI-BLAST search identified a group of approximately 200 MppP-like proteins, most of which are associated with different genomic contexts from SwMppP (i.e. they are not L-End biosynthetic enzymes). We have examined homologs from several of the major clades in this group and found that they seem to fall into one of two classes. About one third of the sequences are adjacent to an NAD(P)H-dependent reductase gene, while the remaining two thirds comprise clades with widely varying genomic contexts. A relatively small number of these sequences are true MppP homologs, occurring in the context of the other L-eEnd biosynthesis genes. We have characterized multiple MppP-like proteins from both classes and found that those co-occurring with the reductase have a subtly different activity, producing 4,5-dehydro-2-ketoarginine. The Ryan group at the University of British Columbia has made the identical observation. For convenience, we call enzymes in this latter group “desaturases”, and those that hydroxylate L-Arg “hydroxylases”. Structure and sequence comparisons suggest that the hydroxylases and desaturases all share essentially identical tertiary structures and active sites. Surprisingly, we have found that the hydroxylase mechanism does not include a 4,5-unsaturated intermediate. Thus, the dehydrated product of the desaturases does not arise from a simple loss of the ability to hydroxylate a common intermediate, but rather from mechanisms that branch from a common intermediate. Structural and kinetics studies of representatives from both classes have suggested several possible explanations for this mechanistic wrinkle, but the more we learn about the structures and substrate-binding modes in these two groups of enzymes, the more similar the hydroxylases and desaturases appear to be.
Sunday
3751828 - Pharmacokinetic strategy for treatment of opioid overdose and addiction
05:25pm - 06:00pm USA / Canada - Central - August 21, 2022 | Location: Great Lakes F (Marriott Marquis Chicago)
Kim Janda, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Opioid use disorders are a global public health concern with less-than-optimal treatment outcomes. In the past year over 15 million Americans have misused opioids. Many of these incidents have been characterized by unintentional poisoning deaths involving synthetic opioids such as fentanyl and carfentanil. The traditional mindset for treating opioid disorders has involved a pharmacodynamic strategy, modulating or disrupting the effects of the drug at sites of action in the body. Unfortunately, this has not yielded broadly effective medications. In view of these limitations, we have investigated a pharmacokinetic strategy. Our approach involves the use of antibodies that target the drug itself to keep the drug below its minimally effective concentration at its sites of action. In this context we will discuss our research program for targeting these synthetic opioids.
Current Topics in Biological Chemistry:
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Phoebe Glazer, Organizer; Ekaterina Pletneva, Organizer
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
3720514 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3725092 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3725745 - Rapamycin-based optical on- and off-switches of protein function | Poster Board #2416
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Rapamycin and its various analogs have been used extensively to induce dimerization of FKBP and FRB domains, as well as proteins fused to them. This has been used to control a wide range of biological processes, including activation of split-proteins, intracellular protein translocation, and protein stabilization. Small molecule induced dimerization of proteins, while an important tool in chemical biology, has previously been limited in its irreversible behavior. Gaining the ability to turn the FKBP-rapamycin-FRB ternary complex back off is significant to the study of the reversible nature of biological processes. In this work, two methods were developed toward this goal – photoswitchable rapamycin analogs were used with the aim of inducing and breaking the ternary complex in response to a configurational change, while ROS-generating rapamycin analogs were used to oxidize and deactivate the ternary complex with singlet oxygen upon irradiation. Though photoswitching did not provide the necessary structural changes required to break a rapamycin-induced ternary complex, we showed targeted protein oxidation via optically triggered singlet oxygen generation can be used as an off-switch for rapamycin-induced protein interactions.
Sunday
3726207 - Design and synthesis of bovine leukemia virus associated peptide based Qβ conjugate eliciting long-lasting immunity in mice | Poster Board #2809
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Bovine leukemia virus (BLV) is a C-type retrovirus of cattle that causes enzootic bovine leukosis with persistent lymphocytosis and lymphoma worldwide with huge economic losses due to reduced milk production and life-expectancy in infected cattle. Currently, no preventative vaccine is commercially available against BLV. Strategies to reduce transmission of this disease, such as segregation and culling of infected animals, are typically not feasible in the US due to significant consequential economic hardships. In this work, we successfully constructed a peptide-conjugate against BLV based on the envelope glycoprotein gp51 peptide-epitope. The antigenic gp51-peptide was covalently linked to a mutant bacteriophage carrier (mQβ) using two different linker strategies, specifically, isothiocyanate and dinitrophenyl adipate. Both vaccine constructs elicited high anti-BLV peptide IgG titers in a mouse model with the isothiocyanate-linker derivatization method being more efficient as it required fewer processing steps. Sera from mice immunized with mQβ-gp51-peptide had significantly higher levels of anti-gp51-peptide IgG antibodies than those from mice injected with gp51-peptide conjugated with a gold standard protein carrier keyhole limpet hemocyanin. Antibodies induced by the mQβ-gp51-peptide construct persisted over 539 days, making it the first BLV peptide-based vaccine candidate to generate such a long-term immunity, an important criterion for an effective vaccine. Post-immune sera from mQβ-gp51 peptide conjugate immunized mice recognized both native gp51 protein and BLV virus particles. Moreover, these sera significantly decreased BLV induced cell-to-cell fusion of susceptible target cells, supporting the high translational potential as a vaccine candidate.
Post-immunization studies for assessment of anti-gp51 antibody immune response in mice.

Post-immunization studies for assessment of anti-gp51 antibody immune response in mice.


Sunday
3726404 - Reactive oxygen species (ROS) mediated prodrugs: mechanism and combination strategy. | Poster Board #2307
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Nitrogen mustard containing compound are known well-known for their high reactivity towards different types of cancers. This reactivity also introduces a higher risk of off-targets and lower selectivity. Using the unique oxidative stress environment of tumors to chemically convert boronic acid derivatives into active nitrogen mustards represents a tissue-specific cancer therapy with reduced adverse effects. Recently, we have reported a potent and selective drug candidate, which is effective in vivo against MDA-MB-468 derived triple negative breast cancer tumor in xenograft mice model. The prodrug FAN-NM-CH3 can be activated in vitro by H2O2 leading to formation of active nitrogen mustards that alkylate DNA. Compound FAN-NM-CH3 was 10 times more cytotoxic than chlorambucil and 16 times more active than melphalan, which are known for their lowest-limiting toxicity within its class of compounds. A combination strategy to enhance H2O2 levels using ascorbic acid was implemented that further improved cytotoxicity 3-folds and lowers drug dose to highest safest dose that inhibits cancer cell growth by 90%. These findings pave the way to investigate the potential of ROS-activated drugs in combination therapy. Although the exact mechanism of its superior activity and selectivity in vivo is yet to be determined. In this work, we measure extracellular H2O2 production level in MDA-MB-468 cells, evaluate effects of ROS quencher/enhancer on prodrug toxicity to define correlation between H2O2 level and efficacy. Additionally understand drug’s mechanism of action as a DNA alkylating agent through evaluation of gene expression.

Sunday
3726786 - Investigating the effects of uracil damage on the therapeutic potential of DNAzymes | Poster Board #2317
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
DNAzymes are catalytically active, single-stranded DNA that coordinate with metal ions to catalyze a number of different reactions in the cell, including mRNA cleavage. This activity makes them an attractive, potential therapeutic for diseases caused by the over expression of specific proteins. However, for DNAzymes to be viable in the cell environment, they must be resistant to chemical insult. This project investigates the consequences of the spontaneous deamination of cytosine on the activity and selectivity of Dz10-23 by replacing such residues in the catalytic core with uracil and observing changes in catalytic activity of mRNA cleavage. Our study demonstrates that, in the presence of a DNA/RNA chimeric substrate with a single RNA base incorporated into the dinucleotide cleavage junction, the Dz10-23 uracil mutants have a reduced activity when compared to the wild type and require the addition of incredibly high concentrations of Mg2+ (500mM) and Mn2+ (10mM). We also show that, when the amount of RNA bases in the substrate is increased, the reactions are extremely accelerated and are efficient at much lower, physiologically relevant metal ion concentrations. Alteration of the substrate RNA content also causes mutations at specific positions to become more or less sensitive to deamination damage. To fully understand the potential impact of DNA damage on mRNA cleavage, we report an exploration of how deamination impacts substrate sequence selectivity. These results indicate that substrate composition plays as equally vital of a role in cleavage efficiency as the DNAzyme core sequence. By comparing our results to the recently determined structure of Dz10-23, we provide new insight into the residues integral for the cleavage reaction and their possible mechanistic roles. The results of this project clearly indicate that the effects of spontaneous deamination of cytosine, and likely other forms of DNA damage, must be considered when designing therapeutic DNAzymes.
Sunday
3727277 - Biocompatible 1,2-carbonyl rearrangements in living E. coli | Poster Board #2511
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Engineered microorganisms are increasingly used in industry as a sustainable source of small molecules. However, native biosynthetic pathways are limited to the generation of molecules already occurring in Nature. One method to introduce this non-native reactivity into a living organism is through the use of abiotic catalysis. By developing and interfacing non-enzymatic biocompatible reactions with microbial metabolism, this could increase the synthetic opportunities provided by biotechnology for the production of value-added compounds.

1,2-carbonyl rearrangements are examples of important functional group transformations not accessed in biology. To address this, we have demonstrated an abiotic 1,2-carbonyl rearrangement in the presence of living Escherichia coli cells - specifically, we observed cell growth of an auxotrophic strain only in the presence of our substrate. Reactivity occurred in M9 growth media, indicating that product formation was dependent on the presence of a biocompatible catalyst. Furthermore, this aqueous rearrangement can interface with enzymatic processes and can be adapted to a range of substrates and products, highlighting how this transformation is applicable to larger, previously inaccessible, biosynthetic pathways.
<b>This work:</b> Cell growth of a auxotrophic strain of <i>E. coli</i> dependent on a biocompatible 1,2-carbonyl rearrangement.

This work: Cell growth of a auxotrophic strain of E. coli dependent on a biocompatible 1,2-carbonyl rearrangement.


Sunday
3728847 - Synthesis and characterization of 2,6-dihydroxypyridine, an intermediate in bacterial nicotinic acid metabolism | Poster Board #2319
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Nicotinic acid (NA) is a common component in many skincare and beauty products, making researchers interested in understanding more about its metabolism and potential impacts to humans and the environment. NA can be degraded by harmless, soil-dwelling bacteria and provides model systems for understanding the metabolism of N-heterocyclic compounds. While the metabolism of NA in Pseudomonas putida and Bordetella bronchiseptica has been determined, the pathway has only been hypothesized within Bacillus niacini. In an effort to confirm this pathway, we are interested in determining the substrate for B. niacini NicC. NicC is hypothesized to be a catalyst in step two of the B. niacini pathway. Potential substrates for the pathway are 2,6-dihydroxypyridine (2,6-DHP), 2,3,6-trihydroxynicotinic acid (2,3,6-THP), or 2,6-dihydroxynicotinic acid (2,6-DHNA). However, these compounds are not commercially available, creating the need for syntheses. This work will highlight the syntheses of these intermediates 2,6-DHP and 2,6-DHNA and assays to determine their ability to act as substrates in NA degradation. By recreating the B. niacini pathway in a laboratory setting, the hope is to understand the mechanisms of this pathway as well as being able to determine societal implications that NA might have.
Sunday
3728934 - Concentration, washing, media exchange of Cart T cells for 5-100 liter bioreactor utilizing single use centrifuges- UFmin and UniFuge. | Poster Board #3018
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
David Richardson , Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Background and Novelty: Collection and washing of Nk T-cells has presented many challenges with
traditional rotor centrifuge methods. The challenges for the bottle centrifuge include product loss -reduced
recovery, lengthy process, and potential for contamination in a open systems. The Single use centrifuge
address all these challenges. The UniFuge was first used for cell collection and vaccine production 12 years
ago. Ten years ago, we started to optimize the collection and washing stratagies of stem cells and
lymphocycytes. Two years ago, we started to collect and wash NK T- cells. These NK T-cells presented
challenges and required additional parameter optimization. In this presentation, we will look at parameters:
feed pump and viability, g force and viabilty, washing volumes, and parameters for overall increased recovery.

Experimental Approach: We will examine the collection and cell washing of NK T-cells in both a small
scale 5 liter bioreactor and in a large Scale 50 liter bioreactor utilizing two different scaled Single Use
Centrifgues- UFmini and UniFuge. Both Single use centrifuges will use the same process parameters scaled
1:5. Overall recovery and viability will be determined by cell counting.

Results and Discussion: Through the optimization of process parameters, we have demonstrated effective
washing of cells, and achieving over all cell recovery greater than 95%.

Sunday
3729306 - GOAT herding: The intracellular trafficking pathway for ghrelin O-acyl transferase | Poster Board #2431
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Ghrelin O-acyltransferase (GOAT) catalyzes the acylation and activation of ghrelin, a peptide hormone involved in a range of physiological pathways and a therapeutic target for metabolism-linked diseases such as obesity and diabetes. GOAT was determined to be localized in the membrane of the endoplasmic reticulum (ER) in early cell-based studies, but recent patient studies detected this integral membrane enzyme in blood plasma and urine. These findings challenge the current understanding of GOAT’s cellular localization and provide a potential mechanism for ghrelin reacylation by plasma membrane-resident GOAT, which would underlie a new limb of the ghrelin signaling pathway within the body.

Our study will determine the cellular distribution of GOAT and intracellular trafficking mechanism governing GOAT localization. Colocalization studies employing immunofluoresence and organellar markers will assign GOAT localization in a panel of cell lines reported to express GOAT at high levels. Used in conjunction with a transiently transfected GFP-tagged GOAT construct, we will determine whether GOAT overexpression leads to altered cellular localization. In addressing the mechanism through which GOAT is intracellularly trafficked, we identified a conserved C-terminal dilysine (KxKxx) sequence in GOAT. This dilysine sequence is a common retrieval motif recognized by coat protein complex I (COPI), which traffics cargo proteins between the Golgi and endoplasmic reticulum (ER) and to the plasma membrane. To investigate whether GOAT is trafficked via a COPI-mediated pathway, we developed a model in which native GOAT could be detected orthogonally to an engineered GOAT construct within a cell. Using this model, we can visualize changes in localization of transgenic GOAT when key lysine residues are replaced with alanine residues compared to native GOAT. These studies are key to advancing our understanding of GOAT, its cellular trafficking, and the impact of cellular localization on ghrelin signaling in different cellular and organismal contexts.

Sunday
3731064 - Enzymatic assay of a potential enzyme to provide insight into the initiation of the biosynthesis of nemamide | Poster Board #2321
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Caenorhabditis elegans is an excellent model organism to study the role of small molecules in biological signaling. Small-molecule pheromones and hormones made by C. elegans influence its development, metabolism, fertility, and life span. Because these signaling molecules are also found in parasitic nematode species, studies of chemical signaling in C. elegans will help us design chemical tools to interfere with the life cycles of parasitic species. Among those signaling molecules, C. elegans produces a hybrid polyketide-nonribosomal peptide, nemamide, which promotes recovery from and survival during starvation-induced larval arrest. Nemamide is biosynthesized by polyketide synthase and nonribosomal synthetase in an assembly line manner. However, why nemamide is synthesized in C. elegans still remains clarified. Investigation on the enzymatic steps of nemamide biosynthesis will elucidate how PKSs and NRPSs contribute to nematode metabolism and how nemamide biosynthesis is regulated. An accessory enzyme ACS-24 is an acyl-CoA synthetase discovered by genome mining, potentially activating fatty acids into fatty-acyl CoA at the beginning of this biosynthetic pathway. To study the catalytic efficiency of ACS-24 for different fatty acids substrates, we tested C7-C11 fatty acids, in particular, several C10 fatty acids with various configurations of double bonds. We found that ACS-24 prefers medium-chain substrates, C9 and C10 fatty acids. Significantly, ACS-24 has a good enzymatic activity for (E)-3-decenoic acid and (Z)-4-decenoic acid. The synthesis of substrates and enzymatic assay results will be presented.

Sunday
3731562 - Investigating the role of pancratistatin in mitochondrial apoptosis via neutron scattering | Poster Board #2904
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Cancer patients are among the most vulnerable in our population with treatment regiments often being highly cytotoxic and harmful, requiring advancements in selective, non-toxic therapeutic alternatives such as Pancratistatin (PST). PST is a natural antiviral alkaloid that has demonstrated specificity towards cancerous cells and explicitly targets the mitochondria. PST initiates apoptosis while leaving healthy, noncancerous cells unscathed and has previously been shown to induce targeted apoptotic action on various human cancer cell lines, such as breast, liver, pancreatic, testicular, and neuroblastoma, while having minimal/ no toxic effects on healthy cell lines. However, due to the lack of experimental evidence for its mechanism of action, the use of PST has yet to be advanced as an efficacious cancer treatment. Above all, the mechanistic pathway must first be elucidated and understood to advance PST as an alternative to the current ineffective treatments available. To investigate this, we use techniques such as use neutron spin-echo (NSE) spectroscopy, very small angle neutron scattering (vSANS), and supporting small angle scattering techniques to study PST’s effect on membrane dynamics using biologically representative model membranes. Our data obtained from NSE suggests that PST stiffens the inner mitochondrial membrane (IMM) mimic by preferentially associating with cardiolipin. Secondly, scattering techniques demonstrate that PST has an ordering effect on the lipids and disrupts their distribution within the IMM, which would interfere with the maintenance and functionality of the active forms of proteins in the electron transport chain. Combined, these results shed light on PST’s effects on the mechanical properties of biomimetic systems with the hopes of advancing PST as an alternative to the current limited treatments available.
Sunday
3732954 - Wireless real-time label free nano-biosensor for bacteria detection | Poster Board #2803
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
An easy access to a simple, rapid, sensitive, and low-cost detector of bacteria is a longstanding demand in not only health care, but also in food industry and homeland security. Currently, molecular (RT-PCR) and cell culture methods that are commonly used for bacteria detection, however, require special reagents, equipment, and training. As a result, these methods are labor intensive, time consuming, and expensive. Here, we report on a novel type of radiofrequency-WiFi-enabled nano-biosensing methodology that, by design, provides real-time wireless monitoring of bacteria at ultralow costs. Currently, commercialized technologies can detect bacterial fragments (e.g. DNA, RNA, protein). In comparison to these existing methods, the new methodology can detect live bacteria directly by monitoring the bacterial metabolism in real-time continuously. In addition to the high sensitivity for detecting 1-10 live bacteria, this disruptive nanosensor is 1) highly specific, 2) palm-size, 3) real-time (3-5 seconds), 4) simple to fabricate and use, 5) wireless in transmitting data, 6) ultralow-cost (<$0.10/each), and 7) highly versatile for detecting bacteria, macrophage, mammalian cells, etc. To the best of our knowledge, no other technology in the market can detect so many different targets. This new work may have fundamental impacts to metabolomics across biology disciplines and to new biosensor innovations and diagnosis advancements in general.
Sunday
3733376 - Tailored vitamin B6- based traps for the in situ detection of hemithioacetal-forming pyridoxal kinases | Poster Board #2805
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The biosynthesis and salvage of pyridoxal phosphate (vitamin B6) which is an essential cofactor in numerous enzymatic reactions, involves pyridoxal kinases (PLK). During their evolution, Gram-positive and Gram-negative bacteria have adopted different active sites that involve not only a catalytic aspartate or cysteine, respectively, but also a common lid cysteine that is contained in a flexible protein loop. This lid cysteine was recently shown to engage with the aldehyde moiety of pyridoxal to form a reversible hemithioacetal that holds the intermediate in place to enable the subsequent phosphorylation reaction. Until now, tools to study the relevance of the lid cysteines in more detail were lacking. We here introduce vitamin B6- based suicide inhibitors with an electrophilic cysteine-reactive capture group to mimic the native hemithioacetal formation. The modification of these traps with an additional alkyne handle at two different positions allows for selective fishing for PLKs in living cells. Importantly, depending on the alkyne attachement site and corresponding protein backbone clashes, we could discriminate between PLKs originating from either Gram-positive or Gram-negative bacteria. Using this approach, we validated the lid cysteine of PLKs as binding sites of our traps not only for previously investigated Gram-positive bacteria, such as S. aureus and E. faecalis, but also in the Gram-negative bacteria E. coli and P. aeruginosa. Furthermore, we unravelled a crucial role of these lid cysteines for catalysis in vitro. In summary, our tailored cofactor mimics enable a reliable readout of lid cysteine-containing PLKs in a variety of organisms, qualifying them as tools for mining further underexplored proteomes for this important enzyme class.
Tailored vitamin B<sub>6</sub>- based traps for the <i>in situ</i> detection of hemithioacetal-forming pyridoxal kinases

Tailored vitamin B6- based traps for the in situ detection of hemithioacetal-forming pyridoxal kinases


Sunday
3733585 - Biogenic Pd nanoparticles enable sustainable C-C cross-coupling reactions and can be interfaced with cellular metabolisms | Poster Board #3006
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Organic chemistry and metabolic engineering are two crucial scientific fields contributing to the production of pharmaceuticals and commodity chemicals. However, merging them together is considered challenging due to the perceived incompatibility of biological and chemical modes of catalysis. Our studies have revealed that palladium (Pd) nanoparticles produced by a sulfate-reducing bacterium Desulfovibrio alaskensis G20 are able to catalyze a number of C-C bond forming cross-coupling reactions under biorelevant conditions (37 °C, aqueous media). The reactivity of these biogenic catalysts was further increased within TPGS designer micelles, which both co-localize reaction components and associate with the bacterial outer membrane. We go on to show that biogenic NPs can be interfaced with H2 production in engineered strains of E. coli to enable one-pot cross-coupling/hydrogenation cascades to access bibenzyl products. Overall, our work highlights the unique reactivity of bacterial metal nanoparticle catalysts and their applications in green chemical synthesis.
Biocompatible one-pot cross-coupling/hydrogenation tandem reactions

Biocompatible one-pot cross-coupling/hydrogenation tandem reactions


Sunday
3734118 - Elucidating the role of zinc in salmon sperm nuclear DNA packaging | Poster Board #2807
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In sperm chromatin, highly basic, positively charged proteins called protamine are used to condense DNA tightly to a final volume roughly 1/20th that of a somatic nucleus. Zinc is present in high concentrations in the seminal fluid and has been shown to play an important role in many sperm functions including sperm chromatin stability, sperm motility, and capacitation. Here, we aim to systematically investigate the role of zinc, and other divalent cations, on the DNA packaging inside sperm nuclei. Divalent cations, such as Zn, are known to effectively screen electrostatic interactions but typically are not able to induce DNA condensation on their own. Counterions having a net charge of +3 or higher are required to overcome the inherently large repulsion between DNA helices and mediate DNA condensation. Using small-angle X-ray scattering (SAXS), we observe that the addition of low concentrations of zinc to isolated salmon sperm nuclei results in tighter DNA packaging. This enhanced DNA packaging is also observed in salmon sperm nuclei with other divalent cations including transition metals, alkaline earth metals, and alkylamines. Experiments with reconstituted protamine/DNA also show the presence of divalents like zinc enhances in vitro condensation through a cooperative attractive interaction. At higher divalent concentrations, we observe a crossover behavior resulting in lower DNA packaging density at high salt concentrations. Lastly, we use ICP-MS to quantify the naturally occurring concentrations of Zinc and other metals in salmon sperm nuclei. Our measurements indicate the use of both protamine and divalent metals may be essential for optimized stabilization of the DNA in sperm chromatin.
Sunday
3734444 - Capillary mediated vitrification: A novel approach for preparing stabilized, ready-to-use reagents | Poster Board #2329
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Although frozen storage is currently the standard practice for storing of biomolecules used as reagents, the process is both inefficient and costly. To ensure stability, reagents must be stored at high concentrations and volumes resulting in significant material discard. While lyophilization can minimize some of these issues; lyophilization requires extensive regent-specific development, may result in loss of yield, and cannot be applied for materials that cannot tolerate freezing. To address these, we have developed a novel and efficient bio-preservation method called capillary-mediated vitrification (CMV). The CMV process is simple to perform at the bench and does not require reagent specific optimization, freezing step and presents minimal risk of matrix interference. The preservation process can be completed in under an hour, producing a stable, dried product. The approach is broadly applicable to different types of biomolecules including enzymes, protein conjugates, antibodies, nucleic acids, small molecules, and viruses. In this report, we used the CMV process to prepare a variety of reagents in a pre-diluted, single use format including: monoclonal antibodies and conjugates, luciferase and luciferin, mRNA and PCR reagents. CMV-preserved alkaline phosphatase and horse radish peroxidase conjugated human IgG antibodies incubated at 55°C overnight showed a similar signal of -20°C stored liquid antibody in an ELISA. The CMV stabilized luciferase and luciferin both maintained >70% activity after storage at 45°C for 6 weeks. A CMV stabilized green fluorescent protein-expressing mRNA stored at 55°C for 100 days had similar transfection efficiency to mRNA that was stored at -80°C prior to use. The CMV-process efficiently preserved PCR reagents which also maintained activity when stored at elevated temperature. In conclusion, CMV is a promising alternative to traditional biopreservation methods and could significantly improve analytical workflows and reduce the need of cold storage for biological molecules used as reagents.
Sunday
3734860 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3734908 - Chemically modified L-nucleic acids for oncogenic microRNA-21 aptamer selection | Poster Board #2331
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Background MicroRNAs (miRs) are known to participate in many important biological processes, and their deregulation is highly related to the pathogenesis of many cancers. One of these, microRNA-21, has been shown to play a pivotal role in carcinogenesis by inhibiting tumor suppressor genes in numerous hematologic and solid tumors. Thereby, a significant amount of effort has been contributed to developing a decent therapeutic tool to target oncogenic miR-21 for clinical diagnosis and treatment. One of the promising approaches is based on nucleic acid aptamer selection via the Systematic Evolution of Ligands by EXponential enrichment (SELEX). A major obstacle impeding the aptamer research field is a relatively short circulating half-live in vivo due to nuclease-mediated degradation. The objective of this project is to select a highly structured and stable in biofluids aptamer to target pathogenic miR-21 based on chemically modified L-type nucleic acids (L-NAs).
Methods 2'-Fluoro and 2'-methoxy derivatized nucleosides and oligonucleotides were synthesized via organic chemistry approaches. The thermal stability assay, crystal structure determination, human serum degradation assay, and circular dichroism studies were performed to explore the effects of chemical modifications in L-NAs.
Results The thermal stability assay and enzymatic assessment reveal that the fluoro-L-oligonucleotides have notable stability. The CD experiment results validate that, the fluoro-modified L-DNA and L-RNA adopt the left-handed helical conformations, which present the mirror-image characteristics of their D-type counterparts. The X-ray crystallographic structural study at the atomic level presents great structural stability of modified L-NAs, as evidenced by the overall and local parameters in the L-helix.
Conclusions Combating the miR-21 molecule by the modified L-RNA binder could potentially provide a promising anticancer drug candidate with enhanced stability and low toxicity. More importantly, the developed strategy based on the chemically modified L-NAs could be an advanced tool for targeting other disease-related RNA fragments.

Sunday
3734943 - Investigating the effect of medium chain triglycerides from vaping oils on pulmonary surfactant systems | Poster Board #2906
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The easy accessibility and growing popularity of vaping has led to an emergence of new users within the past few years. This has led to an outbreak of a relatively new condition known as e-cigarette or vaping use-associated lung injury (EVALI). This syndrome can be caused by interactions of toxins found in vaping solutions with the pulmonary surfactant monolayer in the lungs. Pulmonary surfactant (PS) is responsible for process of respiration, one of the most important physiological functions. Therefore, any impediment into this system that negatively effects the efficacy of this function will have a strong hindrance on the individual and their quality of life. Our group has focused on investigating various toxins found in vaping oils, such as medium chain triglycerides (MCT), and how they affect PS biophysically. One important parameter of investigation is the mechanics of the system, which can provide an idea of how a toxicant can impact the PS system and the associated physiological processes. The technique of neutron spin echo (NSE) allows us to employ neutrons as a means of garnering information of the mechanical properties of membranes, with a resemblance to the composition to human PS. Both in the presence and absence of MCT. A lipid-only PS mimic system (48:32:10:10 DPPC:POPC:POPG:Cholesterol), as well as two animal-derived surfactants, Curosurf (porcine-derived) and BLES (bovine-derived) were investigated in the presence of varying amounts of MCT. This data, as well as supplementary Langmuir trough oscillations studies give an in-depth look at how vaping toxicant MCT affects the biophysical nature of pulmonary surfactant, and will aid in painting a more complete picture on the effects of vaping
Sunday
3734966 - SREBP1 involvement in fatty acid accumulation during necroptosis | Poster Board #2811
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Necroptosis is a type of programmed cell death which is characterized by membrane permeabilization and is known to be associated with strong inflammatory response due to the release of intracellular components based on compromised membrane integrity. Necroptosis is accompanied by key lipid and membrane remodeling and this process is yet to be elucidated. We recently showed that the accumulation of very long chain fatty acids (VLCFAs) contributes to membrane damage during necroptosis. However, the mechanisms that underlie the accumulation of these cytotoxic lipids remain unknown. Using comparative transcriptomics, we found that sterol regulatory element binding protein 1 (SREBP1) and its downstream gene targets are responsible for increased lipid production during necroptosis. SREBP1 activation is triggered via depletion of cholesterol levels at the endoplasmic reticulum is well established. In our work, we show that while the intracellular cholesterol levels remain the same, cholesterol localizes at the plasma membrane, suggesting impaired cholesterol trafficking during necroptosis. We provide evidence that blocking the lysosomal egress of cholesterol activates SREBP1, increases downstream VLCFAs, and induces further toxicity during necroptosis. On the contrary, the inactivation of SREBP1 restores cell viability and membrane integrity during necroptosis. Collectively, our results suggest SREBP1 as a central player in necroptosis that regulate lipid modeling, and as a potential target for therapeutics to reduce membrane permeabilization during necroptosis.
Sunday
3735778 - Total chemical synthesis of Trem-2 ecto domain | Poster Board #2434
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Alzheimer’s disease (AD) is a neurological disorder and considered as the most common cause of dementia. Progression of Alzheimer’s disease leads to detrimental effects on personal wellbeing. Major characteristic histopathological markers of AD are amyloid plaques composed of Amyloid-β (Aβ) peptides and neurofibrillary tangles of phosphorylated tau. There is no cure for this pathological condition. Thus, more research studies should be carried out to understand the molecular level mechanism of the onset of the disease. Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor which is known to activate the signaling pathway of the phagocytosis of cellular debris, lipoproteins, Aβ amyloid aggregates and bacteria. Some mutations such as R47H and R62H within the TREM2 gene are known to increase the risk of AD, and the gene is believed to play a central role in AD pathogenesis. This makes TREM2 receptor is a promising target for therapeutic development to treat AD. However, the molecular level activity of this receptor has not been fully understood yet. We have developed an efficient method for chemically synthesizing ectodomain of TREM2 using solid phase peptide synthesis (SPPS). This methodology will be applied to synthesize TREM2 ectodomain with site-specific modifications. These modifications would include well-defined glycosylation patterns for studying the effect of glycoforms on ligand binding, fluorescence labeling for imaging studies, and biotinylation for chemical library screening. This would allow for better understanding of TREM2, for the use of future therapeutic strategies such as enhancing microglial activation by targeting this receptor.
Sunday
3736758 - Chemical biology approach to uncover influence of sequence variations on CES1 activity in live cells | Poster Board #2333
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Human carboxylesterases (CESs) are serine hydrolases which are responsible for the metabolism of an assortment of ester, amide, thioester, carbonate, and carbamate containing drugs. Given the prevalence of these groups in therapeutics, CESs can metabolize a variety of drugs including remdesivir (Veklury) and oseltamivir (Tamiflu). CES activity is known to be influenced by a variety of factors including single nucleotide polymorphisms (SNPs) and alternative splicing which can produce sequence variants of CESs. These factors can influence drug metabolism by CESs resulting in suboptimal treatments. This is particularly true for one of the two predominant CESs, CES1, where these factors have been linked to adverse clinical outcomes when patients are treated with CES1-substrate therapeutics. Current methods of studying CES1 activity variations caused by SNPs are time consuming and require stable expression of each desired CES1 SNP in human cell lines. This limits the number of SNPs that can be studied at one time. Here we report a fluorescence microscopy-based method that exploits the spatiotemporal nature of this method to enable annotation CES1 sequence variations in human cell lines using transient transfection. This approach can be deployed to rapidly monitor the activity of CES sequence variants. Understanding the activity of CES1 sequence variants could lead to better treatments with CES1-substrate drugs for patient with CES1 SNPs using personalized medicine approaches.
Sunday
3736842 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3736889 - Chemical labeling of endogenou AMPA-type glutamate receptor in live mouse brain | Poster Board #2335
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In the brain, various receptors serve as the starting point for different signal transduction and neural network formation. Among them glutamate receptors have been revealed to be deeply involved in the higher brain functions such as memory and learning. Therefore, it is very important to clarify the function of glutamate receptors. Although various molecular tools have contributed to labeling/imaging of glutamate receptors and regulating their activity, most of them require genetic manipulation valuable methods for Analyzing endogenous receptors are so far very limited. Here we report that endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-activated subtype of glutamate receptors (AMPAR) can be chemically labeled in the live mouse brain using Ligand directed chemistry. Ligand-directed acyl imidazole (LDAI) chemistry, developed by our group, couples selective molecular recognition with proximity-driven chemical reactions to realize specific chemical modification of endogenous target proteins. We herein injected a LDAI reagent for AMPAR into the live mouse brain. The labelling reagent diffused in the brain and was able to specifically modify endogenous AMPARs based on an acyl transfer reaction between amino acid residues of AMPAR surface and acyl imidazole group. The labelling selectivity/efficiency were well characterized by the fluorescence imaging of the brain sections, in gel fluorescence analysis, and immunostaining with related antibodies. Combined with tissue clearing techniques, we have also succeeded in visualizing the 3D localization of endogenous AMPARs in the brain with high spatial resolution.
Sunday
3737263 - Ligand directed protein labeling with tunable Michael acceptors | Poster Board #2415
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Target identification and understanding mechanism of action remains to be one of the biggest challenges in drug discovery pipeline. Photoaffinity labeling has been studied extensively for target identification, but the innate nonspecific protein labeling of the method can lead to false positives. In this study we have explored highly reactive Ligand Directed Tunable Michael acceptors (LDTMAc) with ester functional group. Modular design of LDTMAcs allowed us installation of the ligand on the ester functionality to design them as covalent inhibitors. On the other hand, installation of the ligand on the leaving group resulted in functionality installing probes that leave the active site unoccupied after the labeling reaction. Unlike other ligand directed approaches LDTMAcs’ reactivity is not limited to single amino acid, and they can modify single protein multiple times. These modifications were traced back to multiple histidine, serine, and lysine residues allowing us to map the ligand binding site. Target specificity of LDTMAcs was demonstrated by protein labeling in whole cell lysate. This work suggests the LDTMAcs could be used in identifying targets and binding sites of bio-active compounds.

Sunday
3737392 - Understanding assembly dynamics of the vertebrate lens: an investigation of charge, concentration, and polydispersity on protein homeostasis | Poster Board #2313
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The eye lens is the most protein-rich tissue in the entire body – a feature that is enabled by the precise packing and coordination of varying ratios of multiple crystallin subtypes. Of the three subtypes, β-crystallin is unique, as it exists as a polydisperse, heterogenous distribution of oligomers, which we hypothesize is important for regulating the packing density and optical performance of the lens. However, β-crystallin has historically been difficult to analyze or to ascribe a function to using conventional biochemical or biophysical methods primarily because of its in situ complexity. To address this challenge and test our hypothesis, we utilized size-exclusion chromatography to directly assay the effects of salts on the assembly dynamics of isolated bovine β-crystallin oligomers. We observed that β-crystallin sub-populations persist with distinct sizes and surface charges between 0 and 1M NaCl. When ionic strength and cation valency are further modulated, we observe significant variations in elution profiles that suggest an important role of salt bridges in oligomerization. We then measured how these altered conditions impact protein assembly at the air-liquid interface and observed variations in surface pressure that are dependent on protein subtype. When taken together, our findings elucidate an important role for salt on assembly dynamics that may provide insights for understanding the early development of the lens and the design of improved optical biomaterials.
Sunday
3737449 - Reconstitution of cytoplasmic events of cell-wall recycling from Pseudomonas aeruginosa | Poster Board #2412
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The cell wall, consisting of the crosslinked peptidoglycan (PG), is critical for bacterial viability. Over 50% of the PG is turned over during bacterial growth and division, and its constituents are recycled. The cell wall is broken down in the periplasm by the action of lytic transglycosylases, the reaction products of which are internalized by the permease AmpG. Once in the cytoplasm, the metabolites are processed sequentially by a series of six enzymes to generate a key metabolite on its way to de novo biosynthesis of the peptidoglycan. This work describes the preparations of AmpD, NagZ, AnmK, MupP, AmgK and MurU from Pseudomonas aeruginosa (PAO1). Mass spectrometric assays for each of these enzymes were developed to document the sequential events as a set up to define the metabolic flux for the recycling process. Furthermore, these enzymes would appear to interact with each other in complexes, as defined by surface-plasmon resonance. We will describe these efforts in defining the cytoplasmic events of bacterial cell-wall recycling.
Sunday
3737558 - Uncovering the physiological functions of the thalidomide binding domain of cereblon | Poster Board #2337
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Cereblon (CRBN) is a conserved protein that functions as a substrate recognition adaptor in the CRL4(CRBN) E3 ubiquitin ligase complex. CRBN has attracted growing interest, as it is targeted by the immunomodulatory drugs (IMiDs), therapeutic agents for hematopoietic malignancies, and used as a tool in targeted protein degradation. Moreover, the conservation of CRBN across species and association with neurological development implies its vital biological functions that might be impacted by the IMiDs. However, despite the identification of various neosubstrates recognized by CRBN in presence of the IMiDs, the endogenous substrates and biological functions of CRBN have remained elusive. E3 ubiquitin ligase complexes select proteins for degradation through the recognition of degrons, specific motifs that are sufficient to promote ubiquitylation and degradation when embedded in a substrate. Using integrative approaches including targeted protein degradation, protein engineering and quantitative proteomics, we discovered a degron for the thalidomide binding domain of CRBN that the IMiDs structurally mimic. Here, I will describe the chemical biology approaches to elucidate the novel regulatory process controlled by this degron and the connection of the degron to the physiological function of the thalidomide-binding domain of CRBN and beyond.
Sunday
3737988 - Engineering of diverse post-PKS enzymatic steps in anthracycline biosynthesis | Poster Board #2406
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Streptomyces produce a wide variety of bioactive substances, including the anthracycline anticancer polyketides. Anthracyclines are potent topoisomerase II poisons that inhibit DNA replication in human cancers. Because of their cardiotoxicity, anthracyclines are restricted in their clinical use. However, engineering new anthracycline analogs could decrease cardiotoxicity and could result in more potent antiproliferative activity. Anthracyclines are biosynthesized by a polyketide synthase and are consecutively aromatized, cyclized, and oxidatively modified to afford a tetracyclic anthracycline core. The post polyketide synthase (post-PKS) modification steps include glycosylations, methylations, and oxygenations that generate biological activity. Our team has developed a Streptomyces metabolic engineering platform for interrogation of post-PKS enzymes. Engineering of several constructs for the production of oxidatively modified polyketides was performed using S. coelicolor M1152 delta matAB strains engineered for production of aklavinone, auramycinone, 9-epi-aklavinone, and nogalamycinone. First, we engineered in the aklavinone 11-hydroxylase genes dnrF or rdmE the strains to produce 11-hydroxylated anthracyclinones. Secondly, we engineered in the kstA15/kstA16 or snoaL2/snoaW cassettes for 1-hydroxylation. Thirdly, we engineered in rdmE+rdmC+rdmB to generate 10,11-hydroxylated anthracyclinones. We engineered in a four gene cassette from the kosinostatin pathway consisting of kstA15+kstA16+kstA10+kstA11 cassette to rearrange the alcohol from the 1-postion to the 4-position to generate idarubicin-like derivatives. These proof-of-concept results provide the basis for a comprehensive biosynthetic platform used for synthesis of designer anthracyclines.
Sunday
3738006 - Overexpressing NrfA and its redox partners for mechanistic studies | Poster Board #2436
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Ammonium is an important nitrogen source for plants and microbial communities and is a main component in nitrogen-rich fertilizer. Unfortunately, ammonium can be readily converted to nitrite and nitrate which leach more easily from soils than ammonium. Certain organisms, however, can metabolically utilize nitrate and nitrite and convert them back to ammonium via a pathway known as dissimilatory nitrate reduction to ammonium (DNRA). One of the protein complexes involved in the DNRA pathway is composed of two proteins: NrfH, a membrane-bound quinol oxidase, and its redox partner NrfA, a soluble periplasmic protein that catalyzes the six-electron, eight-proton reduction reaction. This pathway appears to help retain nitrogenous nutrients in the soil, but the mechanistic details involved in this biotic chemical conversion remain largely unknown. Theoretical calculations over the past several years have proved invaluable for predicting the reaction intermediates and helping inform biochemical efforts to unravel the reaction pathway. Our lab has successfully overexpressed and characterized NrfA from Geobacter lovleyi and obtained a crystal structure of this protein. To study the mechanism of nitrite reduction at the NrfA active site in the presence of its physiologically relevant electron donor, we must also overexpress NrfH. NrfH is a membrane bound protein with a soluble, globular head which contains four c-type, bis-His hemes. NrfH receives electrons from the membrane quinol pool and transfers them to NrfA for catalysis. Currently, there is only one crystal structure of NrfH, and this protein has never been successfully overexpressed. Therefore, our lab has created several methods for overexpressing and isolating NrfH. Once NrfH is successfully overexpressed, we will analyze the electron movement from a physiologically relevant redox partner to NrfA, enabling us to better characterize the loading of reducing equivalents to the active site in a more native environment.
Sunday
3738083 - Measuring charge transport properties in RNA oligonucleotides at the single-molecule level | Poster Board #3005
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Oligonucleotides, in particular, RNA have gained importance in the research focus in recent years for several reasons. Not only do several pathogens (e.g., SARS-CoV2) have RNA genomes, but many therapeutic, biotechnological, and modern molecular technologies (e.g., Gene editing & gene silencing) use RNA oligonucleotides. Understanding the electronic properties of RNA is also important for potential applications in medicine, biosensors, and forensics.

Single-molecule electrical techniques have allowed studying the charge transfer process in biomolecules with unprecedented resolution in the last decade. In the biomolecular electronics discipline, the electronic and charge-transport (CT) properties of short oligonucleotides (dsDNA &DNA: RNA hybrid) have been reported extensively. But, despite its biophysical and biological importance, the single-molecule electronic properties of double-stranded (ds)RNA and single-stranded (ss)RNA’s have not been studied.

Here, we use the Scanning tunneling microscope-assisted break junction method (STM-BJ) to study charge transport in short oligonucleotides by obtaining reproducible conductance histograms. We measured, for the first time, the conductance of individual dsRNA molecules and compared it with the conductance of identical DNA: RNA hybrids. The average conductance values are similar for both biomolecules, but the distribution of conductance values shows an order of magnitude higher variability for dsRNA, indicating higher molecular flexibility of dsRNA. In addition, we have measured charge transport in ssRNA oligonucleotides and compared their results with their double-stranded counterpart. The conductance of single-stranded (ssRNA) molecular junctions can be attributed to the base stacking between transient intra-strand base pairs. This result paves the way for measuring different more complex biomolecular interactions at a single-molecule level in the future.
Schematic illustration of the STM-BJ setup.

Schematic illustration of the STM-BJ setup.


Sunday
3738316 - A straightfoward solid-phase enabled strategy to achieve fully functionalized chemoproteomic capture reagents | Poster Board #2408
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Nikolas Burton, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Mass spectrometry-based chemoproteomics has emerged as a powerful technology for functional biology and drug discovery. A central challenge of chemoproteomics is identifying the precise sites of chemical modification on peptides and proteins. A standard chemoproteomics workflow accomplishes this by covalently linking labeled peptides to an enrichment handle, commonly through copper catalyzed azide-alkyne cycloaddition (CuAAC or ‘click’ chemistry). This enrichment handle (e.g. biotin-azide) can then be used to purify the labeled peptides prior to analysis by tandem liquid chromatography mass spectrometry (LC-MS/MS). However, these reagents have some limitations, notably the difficulty in elution of labeled peptides (due to strong biotin-avidin interaction) resulting in poor recovery of labeled peptides as well as false positive identifications due to background labeling. Incorporation of chemical and photocleavable moieties into these reagents help to circumvent these limitations by allowing for facile cleavage of the labeled peptides from the affinity resin. This leads to increased recovery of the labeled peptides while simultaneously excluding non- selectively bound peptides. In addition, the biotin tag is excluded from the sample, reducing the size of the chemical modification and minimizing complicated fragmentation at the MS2 level. The seeming ubiquity of dialkoxydiphenylsilane (DADPS) based cleavable linkers is a testament to their utility for chemoproteomics. DADPS reagents address many of the limitations of other cleavable linkers, including requirements for harsh and contaminating cleavage conditions. This linkage is cleaved under mild, MS-compatible acidic conditions (2-10% formic acid) and has shown to have superior protein and peptide coverage compared to other cleavable linkers. Synthetic strategies for incorporation of DADPS moieties into enrichment reagents remain limited, with previously reported reagents requiring multi-step routes that are hindered by the often-challenging reactions required to form the DADPS linkage. We report the synthesis of a versatile DADPS building block that circumvents many of the aforementioned synthetic challenges. Enabled by the compatibility of this reagent with solid phase synthesis, we developed a straightforward and efficient approach to obtain isotopically differentiated capture reagents that feature a DADPS cleavable linker.
Sunday
3738643 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3738690 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3738772 - Investigation of metal-dependent antimicrobial peptides against C. albicans | Poster Board #2410
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Candida albicans is a commensal fungus that can be found on skin and in the gut microbiota. Though the fungus is ubiquitous, C. albicans infection of the bloodstream can lead to serious illness and death. The rise in drug-resistant Candida infections has led to an urgent need for the development of improved antifungals. We are interested in the use of antimicrobial peptide natural products as fungicidal inhibitors of C. albicans. We have recently found that extracellular concentrations of metal ions can regulate the uptake and potency of some types of antimicrobial peptides. In this work, we have designed a fluorescent antimicrobial peptide probe with photo crosslinking capabilities to investigate the intracellular and extracellular targets of metal-sensitive antimicrobial peptide activity. Through chemical biology and proteomic methods, we aim to identify key proteins involved in metal-dependent peptide trafficking within C. albicans.
Sunday
3738920 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3738985 - Synthesis of mannose-6-phosphonate conjugate for targeted protein degradation through the lysosome | Poster Board #2407
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The binding and tagging of specific protein targets for degradation is a key area of medicinal chemistry and drug development that has become increasingly important as our knowledge of protein/ligand interactions improves. There are several different currently developed routes to targeted protein degradation, mainly through molecular glues, hydrophobic tagging of the target, and heterobifunctional proteolysis targeting chimeras. However, these strategies can only target intracellular proteins. About 40% of proteins in the proteome are outside the cells and many of them are associated with various diseases such as cancer. Lysosome targeting degraders are heterobifunctional molecules that contain a ligand for the protein target to be degraded and a ligand for cell internalization to a lysosomal compartment, which can complement existing strategies for intracellular proteins. One such target for cell internalization is the Mannose-6-Phosphate Receptor (M6PR). Current synthesis of the ligands for M6PR depend on large multivalences in Mannose-6-phosphate (M6P) that require long synthetic paths and are not optimized in their cell internalization. Our goal was to improve upon the synthesis of these lysosome targeting chimeras, and to probe the more exact binding requirements between M6P and its receptor. Our synthetic route for LYTACs streamlines those previously explored for M6P based lysosome targeting degraders and protein degradation studies have shown effective degradation of target proteins at sub micromolar concentrations of the chimera, which validates the refined models.
Sunday
3739207 - BioBricks® platform for engineering of diverse anthracyclinone core scaffolds | Poster Board #2414
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Anthracyclines are aromatic polyketides, or a class of natural products derived from precursor molecules consisting of a chain of alternating ketones and are produced by streptomyces. Anthracyclines are known to be potent therapeutics used for the treatment of human cancers. As a result of toxic side effects, engineering new analogues could decrease toxicity and increase potency allowing safer use. Our approach is to genetically engineer streptomyces to produce these analogues as they are native producers of polyketides. Using these bacteria, we can use enzymes to create the stereocenters needed to produce these compounds.
Anthracyclines are biosynthesized through a multistep enzymatic pathway, which includes a tricyclic intermediate. The biosynthetic route for the transformation of the tricyclic intermediates nogalonic acid or aklanonic acid into tetracyclic anthracyclinones, requires three enzymes. First, nogalonic acid/aklanonic acid undergo O-methylation at the carboxylic acid functionality via nogalonic acid O-methyltranferase to form nogalonic acid methyl ester (NAME) or aklanonic acid methyl ester (AAME), respectively. Second, NAME/AAME undergoes an fourth ring cyclization to form tetracyclic anthracyclinones. Lastly, the anthracyclinone is reduced at C-7 to form nogalamycinone. In this work, we engineered S. coelicolor M1152ΔmatAB strains with different combinations of anthracyclinone biosynthetic genes to produce a comprehensive library of tetracyclic anthracyclinone core scaffolds that are useful for the elaboration of potent anticancer agents.

Sunday
3739359 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3739561 - Novel single-molecule electrical method with applications in Cancer and Covid19 detection | Poster Board #3008
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Cancer is one of the most frequent causes of death globally. Blood samples, or other body fluids, can contain cancer biomarkers such as circulating free tumor nucleic acids (ctNA), promising for early cancer detection in liquid biopsies. Detecting ctNA in the blood is challenging because of the low ctNA concentration and the low frequency of mutations compared to wild-type sequences. Nanotechnology bioelectronics methods can help to address this challenge. In particular, the Scanning Tunneling Microscopic (STM)-assisted break junctions method (STM-BJ) has recently allowed the first demonstration of detection and identification of RNA from E.Coli via single-molecule conductance. This is an ideal emerging technique for liquid biopsy bioelectronics since it is extremely sensitive, specific, and non-invasive.
This work focuses on characterizing ctNAs using the STM-BJ to investigate an effective method for their ultra-sensitive detection in complex samples. The study's central hypothesis is that the sequences of ctNAs can be used to detect cancers by finding their unique electronic fingerprints. We focus the study on KRAS, BRAF, and NRAS as effective cancer biomarkers. We have already obtained preliminary data for wild-type and mutated RNA sequences for a few candidate cancer biomarkers (Ex: KRAS Exon 2 Wild type, G12V, and G12C mutations). Our initial analysis and the results pave the way for the early detection of bioelectronics fingerprints from biomarkers through liquid biopsy using nanotechnology. The same idea can be applied to other applications such as covid detection. We conducted a comprehensive literature survey and bioinformatics analysis to identify the most appropriate candidate nucleic acid sequences for all human coronaviruses, SARS-Cov2, and other SARS-Cov2 variants such as Delta, Omicron, etc and obtained some preliminary single-molecule conductance data. This emerging method may allow beginning treatments early, potentially saving many lives from cancer and covid patients in the future.
Figure 1: Electrical cancer detection via single-molecule techniques in liquid biopsies

Figure 1: Electrical cancer detection via single-molecule techniques in liquid biopsies


Sunday
3739654 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3739712 - Selection of GDP-fucose by active site residues in Arabidopsis thaliana SPY | Poster Board #2418
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) transfers O-GlcNAc from the nucleotide-sugar UDP-GlcNAc to thousands of nuclear and cytoplasmic proteins. OGT is an essential enzyme, and misregulation of O-GlcNAcylation has been linked to cancers and
neurodegeneration. Most eukaryotic organisms possess a single structural homolog of OGT. However, Arabidopsis thaliana has two: Spindly (SPY) and Secret Agent (SEC). Whilst being structurally similar, SPY and SEC select different nucleotide-sugars, GDP-fucose and UDP-GlcNAc, respectively. Using human OGT’s crystal structure, we made corresponding active site mutations in AtSPY that we hypothesize are critical for GDP-fucose binding and glycosyltransferase activity. Here, we recombinantly expressed each AtSPY mutant in E. coli and purified them using a two-step purification method. The catalytic activity of each mutant was tested using an in vitro malachite green glycosyltransferase assay to determine whether OGT’s catalysis mechanism is conserved in AtSPY and identify the active site residues that govern GDP-fucose selection.

Sunday
3739736 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3739752 - Intracellular protein targets of opioid drugs | Poster Board #2428
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The opioids possess analgesic properties, yet unlike the endogenous opioid peptides, exogenous opioids have dangerous effects on multiple organ systems, including the respiratory, cardiovascular, and musculoskeletal systems, which combined with their addictive effects can lead to death. These promiscuous properties suggest that exogenous opioids engage a broader array of targets within the cellular proteome beyond the opioid receptors. To directly investigate the cellular targets of exogenous opioids, we developed three probes with specific chemical functionalization to enable the unbiased detection and visualization of these protein interactions; herein, photo-click-morphine (PCM), enone-morphine (EnM), and di-alkynyl-acetyl-morphine (DAAM). We developed PCM and EnM to capture morphine binding sites and DAAM for profiling acetylation sites that arise specifically from opioid treatment in the cell. We employed these three probes within a chemical proteomics platform to characterize protein interaction partners of the opioids throughout the cell. This platform involves (1) treatment of cells or an animal model with the opioid probe, (2) chemical enrichment of the protein binding partners or acetylation sites, and (3) unbiased identification of the protein targets and modification sites by mass spectrometry. Application of this chemical proteomics platform to SH-SY5Y cells with DAAM revealed the engagement and acetylation of a novel target, the mitochondrial phosphate carrier protein or PiC (SLC25A3). PiC is a ubiquitously expressed and essential transporter of phosphate and copper ions from the cytosol to the mitochondrial matrix. The physiological role of PiC in regulating oxidative phosphorylation and pH is necessary for normal cardiac and muscular function and is associated with roles in other tissues. The effect of exogenous opioid binding and acetylation of PiC on mitochondrial function in neuronal and cardiac cells will be presented. These findings provide novel targets that may have a relationship to the side effects of opioid drugs while expanding the chemical toolbox for studying the mechanisms of these molecules.
Sunday
3739834 - Analysis of bioactive molecules produced by fungus-growing ants' bacterial symbionts in an unexplored desert environment | Poster Board #2420
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
There is an urgent and growing need for novel antibiotics with the rapid emergence of antibiotic-resistant pathogens. Ecologically-guided discovery is a strategy that focuses on how and where specially evolved metabolites such as antibacterials and antifungals are used in natural settings. This strategy reveals unique molecules with potential for use as antibiotics. An unexplored niche with great potential for antibiotic discovery exists with Southwestern fungus-farming Trachymyrmex ants and their Actinobacteria symbionts. In exchange for nutrients from the ant, the ants’ bacterial symbiont produces antifungal molecules that protect the ants’ fungal cultivar from invasion by pathogenic microorganisms and antibacterial molecules to protect themselves from niche competitors. Recently, new antibiotics have been isolated from Trachymyrmex ant symbionts collected in the southwestern U.S., such as a novel analog of the antibiotic nocamycin. This finding suggests that this unique environment may yield additional novel metabolites. Here we report preliminary findings from a large-scale and systematic analysis of symbiont-derived natural products from this niche to understand their distribution and discovery potential, with the ultimate goal of discovering and characterizing novel bioactive compounds. We have assembled a collection of Actinobacteria isolated from 40 colonies of Trachymyrmex ants in Arizona. An extract library has been prepared from this collection, and ongoing metabolomics analysis and antibiotic activity testing of that library indicates the distribution of active molecules.
Sunday
3740095 - Identification of an antibiotic produced by ant-associated amycolatopsis bacteria | Poster Board #2426
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Many antibiotics are derived from natural environments, but we know little about the ecological function of most of these molecules. Trachymyrmex ants of the American Southwest and their microbial associates form an ideal system for interrogating antibiotic function in nature. The fungus-growing ants in this niche are postulated to participate in a mutualistic relationship in which bacterial symbionts protect the ants’ fungal cultivar from pathogenic fungi in return for nutrients from the ants. Amycolatopsis strain 17SM-8, isolated from the cuticle of the ant Trachymyrmex smithi, antagonizes the fungal pathogen of the ants’ colonies, and we wanted to understand the chemical basis for that antagonism. Using bioassay guided fractionation, we have established that the molecule responsible is the antibiotic ECO-0501. We have confirmed the identity of the molecule by comparison to an authentic standard using LC-MS/MS. This molecule’s antibacterial activity had previously been documented, but this is the first report to our knowledge of its antifungal activity. The discovery of ECO-0501, an antibiotic that previously had no known ecological function, suggests Amycolatopsis as an effective defensive microbial associate.
Sunday
3740370 - Biomimetic model of photosynthetic light harvesting: Templating chlorophyll arrays within a self-assembling protein capsid | Poster Board #2430
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Deborah Zhuang, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In natural photosynthetic systems, energy absorbed from incident light is efficiently transferred across numerous chromophores in rigid protein scaffolds to the reaction center, where a charge separation event occurs to ultimately produce ATP. However, a full and detailed understanding of these systems has remained elusive, largely due to the intricacy of their chromophore networks and instability of the natural proteins to mutation. Self-assembling viral protein capsids have provided convenient scaffolds for the construction of many new materials with well-defined nanoscale architectures, and readily assemble into highly symmetric, rigid shapes ideal for biomimetic light-harvesting models. The tobacco mosaic virus (TMV) coat protein is a highly mutable protein that assembles into a double-disk structure containing a C2 axis of symmetry and presents many sites at which functional residue mutations can be introduced.
We have expressed a genetic fusion of two TMV monomers still properly assembles into a double-layered disk structure in which we can now asymmetrically modify the top and bottom disks. A non-native histidine coordination site was introduced to the gap region between the two disks in this new system to bind the magnesium center of chlorophyllide, a water-soluble precursor to chlorophyll. This variant serves to template a circular array of protein-embedded pigments as a minimal model of the bioenergetic processes found in nature. Photophysical characterization of isolated chlorophyllides embedded within the TMV complexes reveal that these pigments have an extended fluorescence lifetime compared to that of the free pigment. This may contribute to the rapid rates of interchromophore energy transfer that were observed within the fully-labeled chromophore array. By introducing mutations to the sites surrounding the chromophore within the binding pocket through both experimental and computational efforts, we then examined the effects of the protein microenvironment on the chromophore’s excited state dynamics and energy transfer rates. These results will provide insight into interactions that could lead to increased light-harvesting efficiency through slowing of the nonradiative relaxation pathways. In all, this system provides a clear and effective path to producing highly controllable artificial light-harvesting systems that can increasingly mimic their natural counterparts, thus aiding to elucidate natural photosynthetic mechanisms.

Sunday
3740418 - Cisplatin binding causes RNA destabilization and inhibition of a CRISPR model system | Poster Board #2432
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
With the advent of CRISPR gene editing and the ongoing COVID-19 pandemic, RNA has received enormous attention as a biomolecule in the fields of medicine, public health, and biotechnology. Over the last decade, various types of RNA molecules have been exploited as valuable drug targets by different classes of pharmaceutical agents in combating infections and diseases. In this work, we have investigated the binding of Cisplatin with two different RNA molecules. UV melting and Isothermal Titration Calorimetry studies reveal that Cisplatin binding leads to destabilization of short duplex RNA likely due to cisplatin-RNA adduct formation. We have also successfully developed an in vitro CRISPR-Cas9 DNA cleavage model system in our laboratory. Gel electrophoretic analysis using this model system shows that Cisplatin binding to guide RNA leads to a concentration-dependent reduction in Cas9-mediated DNA cleavage.
Sunday
3740589 - Multidisciplinary materials discovery | Poster Board #2821
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Alan Aspuru-Guzik, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In this talk I will talk about the opportunities and challenges of carrying multidisciplinary research in materials science.
Sunday
3740682 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3740737 - Activation mechanism of the human smoothened receptor | Poster Board #2401
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Smoothened (SMO) is a membrane protein of the Class F subfamily of G-Protein Coupled Receptors (GPCRs) and maintains homeostasis of cellular differentiation. SMO undergoes activation, transmitting signals across the membrane making it amenable to bind to its intracellular binding partner. Overstimulation of SMO has been linked to medulloblastoma and basal cell carcinoma. Vismodegib and Sonidegib are currently two FDA-approved SMO antagonists but suffer from chemoresistance. SMO bound to agonists and antagonists at sites in the Transmembrane Domain (TMD) and the Cysteine Rich Domain (CRD) have been characterized. The endogenous agonist of SMO, cholesterol, is hypothesized to travel through a hydrophobic tunnel inside SMO to activate it. Receptor activation has been studied at length for Class A receptors, but the details of Class F receptor activation remain unknown. While crystal structures of the inactive and active SMO outline the residue-level transitions, a kinetic view of the overall activation process remains unexplored for Class F receptors.

We performed ~250µs of molecular dynamics simulations, to describe SMO's activation process in atomistic detail. A molecular switch, conserved across Class F and analogous to the activation-mediating D-R-Y motif in Class A receptors, is observed to break during activation. We also show that this transition occurs in a stage-wise movement of the transmembrane helices - TM6 first, followed by TM5. To see how modulators affect SMO activity, we simulated agonist and antagonist-bound SMO. We observed that agonist-bound SMO has an expanded hydrophobic tunnel in SMO's core TMD, while antagonist-bound SMO shrinks this tunnel. In summary, this study establishes links between activation of Class F and Class A GPCRs and shows that SMO's activation process rearranges the core transmembrane domain to open a hydrophobic conduit.

Sunday
3740824 - Mapping substrate specificity of 3-chymotrypsin-like proteases using high-throughput experimental screening and computational modeling | Poster Board #2520
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
3C and 3C-like proteases (formally known as 3-chymotrypsin-like proteases) from RNA viruses belong to the chymotrypsin-like fold and are highly specific in cleaving the viral polyprotein. Mapping of substrate specificity of 3C-like proteases of different substrate specificities is significant since it will develop a novel methodological framework combining computation and experiments for the prediction, measurement, and design of protease specificity landscapes.
From a curated dataset of (~2000) 3C-like proteases belonging to various viruses with known canonical substrates, we focused on a subset of these proteins (~25-30) with well-characterized structures. Then, we classified these proteases into three different specificity groups according to their preference for sidechains at the scissile bond (i.e., P1 and P1’ positions) of their canonical substrate sequences.
We selected 5 proteases each for their specificity towards Q/X, R/X and E/X respectively (P1/P1’; X = A/S/G). Within each of these three specificity classes, each protease is part of a different virus and their active site residue sequence homology ranges from 20%- 100% while overall sequence similarity is on average ~50%. Thus, the choice of benchmark cases enabled sampling diverse active site environments while maintaining the overall fold and P1/P1’ recognition.
First, we validated the native cleavage activity of the proteases using the Yeast Endosomal Sequestration Screening (YESS) cleavage assay. Then, for each protease, we experimentally sampled ~1 million different substrate combinations through the construction of a substrate library exhibiting NNK codon degeneracy P6 through P2 positions (5 residues) of each sequence. Fluorescence activated cell sorting (FACS) followed by next-generation sequencing enabled collection of cleaved, uncleaved and partially cleaved sequences for each protease. Structural modeling of these protein-peptide complexes provided us with thousands of data points for input into a graph convolutional neural network that will enable prediction of protease specificity landscapes.

Sunday
3740834 - Novel lead compounds demonstrating anti-biofilm properties in Staphylococcus aureus may act as Nor-A efflux pump inhibitors. | Poster Board #2403
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Numerous studies have shown that bacteria quantify their density and collectively carry out a certain response with a communication mechanism known as quorum sensing. Biofilms are a type of virulence factor that forms once a bacterial quorum is reached within the host, preventing the host’s immune system from detecting and ultimately eradicating these pathogenic bacteria. Efflux pumps may also be involved in biofilm production. These pumps are transport proteins that are responsible for the extrusion of various substances, including toxins, quorum sensing molecules, and biofilm components. The increased activity of efflux pumps has allowed antibiotic-resistant bacteria to increase their survival rates. This study tests certain drugs that were designed to resemble quorum sensing signaling molecules to inhibit biofilm formation. Since these drugs also have structural homology with known Nor-A efflux pump inhibitors (EPI) in S. aureus, we tested their activity on this pump. Fourteen of our previously synthesized drugs share this structural homology, with five of them revealing significant biofilm reduction in SA-1199 (wildtype) and SA-1199B (Nor-A overexpressor) but not in SA-K1758 (Nor-A knockout). This indicates that the drugs function in targeting the Nor-A pump rather than as competitive inhibitors at the receptor sites of the S. aureus. The conjugated aromatic ring with alternating electron-withdrawing and donating groups on the ortho and para positions appear to affect the inhibitory characteristics of these drugs. Developing a cocktail of drugs that curtail quorum sensing and inhibit efflux pump activity can maximize biofilm inhibition in S. aureus.

Sunday
3740883 - Viperin: A novel antiviral protein | Poster Board #3000
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum associated, INterferon inducible) is a recently recognized human radical S-adenosylmethionine (SAM) enzyme involved in cellular antiviral response. Radical SAM enzymes use SAM as a cofactor to generate the reactive 5’-deoxyadenosyl radical (5’dAdo) and all are very oxygen-sensitive. Viperin is conserved across all kingdoms of life, indicating its ancient and important role in the antiviral response. In animals, viperin functions in two ways: it synthesizes an antiviral nucleotide, 3’-deoxy-3’,4’-didehydro-CTP (ddhCTP) from cytidine triphosphate (CTP), that inhibits replication of some RNA viruses by acting as a viral polymerase chain terminator; it also acts as an important regulator of the antiviral response through interactions with numerous cellular and viral proteins.
We recently used proteomic analysis to investigate the interactome of viperin. Multiple cholesterol biosynthetic enzymes were among the most enriched proteins, with lanosterol synthase (LS) and squalene monooxygenase (SM) being the top two hits. Many enveloped viruses require cholesterol-rich membranes to successfully bud from the cell and we have shown that over-expression of viperin inhibits cholesterol biosynthesis. Studies in our lab are directed towards understanding the regulatory effect on cholesterol biosynthesis that viperin may exert through its interactions with SM and LS.
The proteomic analysis also identified several ubiquitin-conjugating enzymes in viperin’s interactome; indeed, viperin is known to promote the ubiquitin-dependent degradation of various cellular and viral proteins. Viperin also potentiates innate immune signaling pathway by activating the E3 ubiquitin ligase, TRAF6 to polyubiquitinate IRAK1. We have reconstituted the viperin-TRAF6 interaction in vitro to directly demonstrate viperin’s role in activating the innate immune signaling pathway. Our studies also implying a broader role for viperin as an activator of protein ubiquitination leading to proteasomal degradation pathway of its target proteins.

Sunday
3740885 - Biophysical insights into the regulation of the tyrosine phosphatase PTP1B by the adaptor protein Grb2 | Poster Board #2510
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Tyrosine phosphatases are important signaling enzymes that help regulate the phosphorylation states of proteins. Their function is critical to cell communication, proliferation, and migration. Impaired activity of these enzymes can cause altered levels of tyrosine phosphorylation, which can lead to human disease. Protein tyrosine phosphatase 1B (PTP1B) is most well-known for its role in the insulin and leptin receptor pathways, making it an attractive target for the treatment of type II diabetes and obesity. PTP1B has been shown to interact with the adaptor protein Grb2, which is a critical mediator between cell membrane-bound growth factor receptors and the Ras signaling pathway. Grb2 also interacts with insulin receptor substrate 1, a known substrate of PTP1B. We have confirmed the interaction between PTP1B and Grb2, which is driven in part by the proline-rich region of PTP1B. Additionally, we have shown that Grb2 binding to PTP1B causes an increase in its catalytic activity. Interestingly, this effect does not appear to be dependent on the proline-rich region of PTP1B, which may suggest a secondary contact region between the two proteins. Finally, we are using biophysical techniques such as NMR and HDX-MS to further characterize this interaction, in order to determine the molecular basis for the effect of Grb2 binding on PTP1B activity. A better understanding of this interaction will reveal new insights into PTP1B and Grb2 signaling biology.
Sunday
3740956 - Designing chemoproteomic probes for activity-based profiling of tyrosine phosphatases | Poster Board #2421
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Tyrosine phosphorylation is an important post-translational modification that can modulate protein-protein interactions, protein localization, stability, and enzyme activity. Many diseases including cancer arise from the dysregulation of tyrosine phosphorylation. This post-translational modification is mediated by tyrosine kinases, which phosphorylate proteins, and tyrosine phosphatases, which dephosphorylate proteins. While many kinases are well-characterized at the level of structure and signaling, relatively little is known about the regulation of protein tyrosine phosphatases (PTPs). As PTPs can function both as oncogenes and tumor suppressors, understanding their function and regulation will be crucial in developing new cancer treatments and identifying new drug targets. Thus, there is demand for the development of new chemical tools to monitor PTP activity in different cell signaling events.

We are developing covalent ligands that can be used to report on the activity of PTPs across the proteome. To design these activity-based probes, we first surveyed a broad array of scaffolds and thiol-reactive electrophilic warheads. Candidate compounds with bromoacetamide and chloroacetamide warheads were synthesized and characterized using various reactivity and enzyme inhibition assays. Based on these assays, a few candidates that displayed the desired characteristics were augmented with a bio-orthogonal alkyne tag to facilitate downstream enrichment for proteomics experiments. The proteome-wide reactivity, click-chemistry compatibility, and cell permeability of these molecules were examined in HEK293 cells. We then assessed the proteome-wide selectivity of these probes in HEK293 and Jurkat T cells by enriching labeled proteins and conducting mass spectrometry proteomics. Guided by these experiments, we are examining how PTP labeling by our top-performing probes changes as a function of the activation of Jurkat T cells, as PTPs play an important role in T cell signaling. We anticipate that changes in PTP labeling by our probes will correlate with changes in the activity of those PTPs. Ultimately, we anticipate these probes will be used to investigate and discover new regulatory mechanisms of PTPs in signal transduction pathways.

Sunday
3741302 - Binding small molecules to the zinc(II) dependent transcription factor AdcR | Poster Board #2405
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Zinc(II) is a micronutrient required by all known life. In both prokaryotic and eukaryotic organisms, intracellular zinc(II) concentrations are controlled by a family of transcription factors that regulate metal uptake and efflux systems. In S. pneumoniae, two proteins have been found to be responsible for Zn2+ homeostasis, namely AdcR and SczA. These proteins share little structural homology, but are both Zn2+ dependent transcription factors, where AdcR regulates Zn2+ uptake and SczA controls Zn2+ efflux. AdcR is a member of the MarR family of transcription factors and directly represses adcRCBA, adcAII-phtD, phtA, phtB, and phtE genes in the presence of Zn2+. AdcR is a dimer and contains two Zn2+ ions per monomeric unit, one of which has a water molecule in its r coordination sphere. Here we report our initial efforts to displace this water molecule with small molecules known to coordinate to zinc(II) ions in vitro and in vivo. A family of phenylsulfonamides have shown promise as AdcR binding molecules. Full characterization of the thermodynamic parameters for each of these binding events were measured using common biophysical techniques, which allow some insight into how the structure of these impact efficiency. Each of the AdcR/phenylsulfonamide complexes was screened against the AdcR binding domain to determine if these small molecules can disrupt the equilibria required for zinc(II) homeostasis. The antimicrobial activity of these agents was also monitored against laboratory strains of S. pneumoniae.
Sunday
3741437 - Deciphering stress signaling in the plant chemistry of Maize | Poster Board #3004
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In nature, plants encounter multiple stress factors, yet, to date, our understanding of how plants actively respond to combinatorial stress remains limited. Critically understanding how plants cope with combinatorial stress can allow us to mitigate their response through sustainable crop production and breeding efforts. Plant responses to simultaneous abiotic and biotic stress factors involve changes in plant chemistry. These changes alter the blend of secondary compounds produced and subsequently the volatile organic compounds emitted. This study aims to evaluate the interactive responses to the combinatorial stress of elevated carbon dioxide, flooding, and herbivory on Maize grown in environmental growth chambers. Maize plants were grown for 28 days in ambient or elevated atmospheric carbon, and in this atmosphere, subjected to flooding and herbivory. Volatile organic compounds were collected using Solid Phase Microextraction and analyzed using Gas Chromatography coupled with Mass Spectrometry. Preliminary results indicate that the interactive stress of elevated carbon dioxide, flooding, and herbivory alter plant chemistry. In addition, the compounds identified are known to be herbivore induced and environmental stress induced. This suggests that combinatorial stress invokes potentially additive phytochemical stress responses and reinforces the need for sustainable crop production.

Sunday
3741493 - Insights into bacterial membrane protein quality control: YccA and its interaction with FtsH | Poster Board #2917
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Membrane proteins present more than 25% of the proteome of all cells, and mediate the cell's interaction with its environment. They are of prime pharmaceutical interest as they constitute ~50% of known and potential new drug targets. Understanding the function and molecular structure of this class of proteins is of key interest. A large proportion of proteins needs to be incorporated into specific cellular membranes; in E. coli, it is about 20-30% of the total number. This process is known as protein translocation. Several membrane proteins are involved in protein quality control mechanisms during this process. FtsH is a conserved bacterial protease that degrades improperly folded integral membrane proteins using an ATP-dependent mechanism. An integral membrane protein, YccA, comprising seven transmembrane regions, is one of the substrates of FtsH and inhibits the FtsH proteolytic activity. SecY is degraded by FtsH when it is not in a complex with SecE and SecG. The membrane protein YccA, inhibits FtsH, and counteracts this destruction, when it is overproduced or stabilized. In the mutant form of YccA lacking eight amino acids in the N-terminal end, SecY was found to be stable. This mutant protein, YccA11, binds to FtsH, but cannot be degraded by FtsH, unlike wild-type YccA. In this study, we cloned both YccA and YccA11 from the E. coli K-12 strain genomic DNA using Gibson assembly method with a His-tag on the N-terminal end, and overexpressed in E. coli. Membranes were solubilized with either DDM or LMNG. Proteins were purified by affinity chromatography. The size and purity of YccA was confirmed by SDS-PAGE, and size exclusion chromatography. The stability of YccA was investigated by cross-linking with BS3 (bis(sulfosuccinimidyl)suberate), also its interaction with FtsH was investigated using cross-linking mass spectrometry with DSBU (Disuccinimidyl Dibutyric Urea). Furthermore, the degradation of YccA was studied by the recombinant FtsH. The outcomes will help structural studies and understanding of the mechanism behind this fundamental process.
Sunday
3741515 - Distinguishing the molecular contribution from Indirect communications by 3D breast cancer models with temporal heterogeneity control | Poster Board #2409
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Intratumor heterogeneity in breast cancer is a significant hindrance to treatment as it promotes drug resistance and cancer progression, as well as increases the risk of disease relapse. Numerous studies have reported genetic differences during breast cancer development. However, there is a lack of understanding of the relationship between tumour composition and temporal heterogeneity in a tumour at the molecular phenotypic level. This study aims to identify how the increasing level of malignancy in breast cancer induces the epithelial-mesenchymal transition of non-tumorigenic cells. Two models were used to study indirect communications between cells by (1) extracellular vesicles (EVs) and (2) overall cellular secretions. MCF10AT, an isogenic breast epithelial cell line series, which includes four cell lines representing normal to fully malignant states (M1-M4), was used for this study. The non-tumorigenic cells (M1) were first exposed to isolated EVs from (pre-)malignant cells (M2 to M4). Then, M2 to M4 cells were seeded separately on a transwell membrane that allows secretions from the top layer to reach the bottom layer of M1 cells without physical contact between cell types. To visualize EV uptake by cells, Raman spectroscopic imaging, a label-free technique, was used. The EV and cell secretion exposed M1 cells were grown in a 3D manner to assess the malignant transition of M1 cells. Morphological and molecular changes in the M1 cells were analysed through acini development. The effect of EVs and that of the remaining secretions were probed through different degrees of exposure. This work forms a model of indirect communications between normal and cancerous cells to predict temporal heterogeneity. Establishing a clear relationship between tumour composition and temporal heterogeneity is useful for both prognosis and development of targeted and patient-specific treatments.
Sunday
3741527 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3741567 - Thermodynamic and structural characterization of the ferritin-NCOA4 complex | Poster Board #2411
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The iron storage protein ferritin is a highly conserved supramolecular nanostructure that plays a crucial role in iron homeostasis. Eukaryotic ferritins are predominantly heteropolymeric species consisting of two similar but functionally different subunit types, named H and L. The two subunits co-assemble in various H to L ratios with tissue-specific distributions to form shell-like protein structures of 24 subunits (isoferritins) within which a mineralized iron core is stored. Recent studies showed the existence of a specific ferritin degradation mechanism, called ferritinophagy, facilitated by the nuclear receptor coactivator-4 (NCOA4), which under iron deplete conditions binds to ferritin and carries it to the auto phagolysosomes for iron release. Here we have examined the complexation reaction between NCOA4 and different isoferritins using isothermal titration calorimetry (ITC), mass spectrometry (MS), electrophoretic mobility shift assay (EMSA), and western blot. Our ITC data revealed a molecular association that is both enthalpically and entropically favored, with a binding stoichiometry of ~ 8 NCOA4 fragments per ferritin shell and a dissociation constant of ~ 0.4 uM for the homopolymer H-ferritin. EMSA analysis showed a clear band mobility shift that saturates at ~ 8:1 NCOA1:ferritin ratio and the complexation reaction was confirmed by western blot and MS experiments. Preliminary cryogenic electron microscopy (Cryo-EM) experiments showed a structure with NCOA4 molecules bound at or near the eight hydrophilic 3-fold channels of ferritin
Sunday
3741595 - Development of biosensors specific for free NEDD8 | Poster Board #2419
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
NEDD8 is a small protein involved in essential pathways including cell cycle regulation and DNA-damage-induced apoptosis. It regulates these functions through conjugation to other proteins. In living cells, NEDD8 exists in 3 forms (pools): Conjugated NEDD8 (NEDD8 conjugated to some other protein, of which there are many), Activated NEDD8 (NEDD8 conjugated to NEDD8 enzymes), and Free NEDD8 (unconjugated NEDD8). These pools’ concentrations are kept in balance by neddylation, the conjugation of free NEDD8 to a protein, deneddylation, the separation of conjugated NEDD8 from its target to make free NEDD8, and translation of the NEDD8 gene to manufacture more NEDD8.
Maintaining the balance of the two NEDD8 pools is critical to maintaining homeostasis. Chemicals that inhibit neddylating or deneddylating enzymes can potentially be used to treat diseases caused by overexpression of these enzymes. Current NEDD8-related enzyme assays are specialized for NEDD8 conjugated to a specific target and cannot be efficiently scaled up or adapted to be used with other forms of conjugated NEDD8. There is currently no sensor that can detect free NEDD8. Because free NEDD8 is either a substrate or product of all NEDD8-related enzymes, development of such a sensor would enable the study of the ratio between the two NEDD8 pools in cells and enable large-scale drug screening. Our project aims to develop a sensor to measure free NEDD8 concentrations for general NEDD8-related enzyme assays. Such a sensor should have high sensitivity and specificity to NEDD8 and must be equipped with a method for signaling when it has bound to the target.
To this end, we are developing a fluorescent biosensor that can be used to quantify the concentration of free NEDD8 in-vitro and in living cells. Our sensor is derived from a genetically modified deneddylase enzyme. It is manufactured by transforming E. coli cells with a cloned plasmid with the gene encoding our sensor. Our current work focusses on optimizing the sensitivity and selectivity of our sensor.
We have produced several models of our sensor with different signaling methods and active site mutations. We have characterized one version of the sensor using a binding assay. Its Kd for free NEDD8 is 212 nM, and its Kd for conjugated NEDD8 is 140 μM. These data show us that our sensor has 660 times higher affinity for free NEDD8 than for conjugated NEDD8. Future work will focus on applying the sensor to free NEDD8 assays and drug screening.

Sunday
3741598 - Development of chemical tools for deep tissue imaging of calcium | Poster Board #2614
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Metal ions such as calcium (Ca2+) are critical to the survival of all living organisms as it controls the contraction of muscles, coagulation of blood, and neurotransmitter release. Because of its versatile nature, Ca2+ is one of the most highly regulated metal ions in the body. Current optical imaging methods to monitor Ca2+ levels in vivo are limited by depth penetration and attenuation by incident light. Furthermore, in the context of studying Ca2+ fluctuation in the brain, intravital microscopy is utilized which is a highly invasive procedure. Photoacoustic imaging (PA) and the application of activatable probes have been developed to circumvent these issues. PA probes take advantage of the photoacoustic effect in which light is transformed into heat that can then be detected as ultrasound waves. However, Ca2+ responsive PA probes have not been studied in the context of deep tissue imaging. In this work, we have tuned various dye platforms that have improved sensitivity and depth penetration to investigate the role of Ca2+ in relation to various diseases such as ovarian insufficiency and Alzheimer’s Disease.
Sunday
3741601 - Expression and purification of the full length nuclear receptor coactivator-4 (NCOA4) | Poster Board #2413
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Nuclear receptor coactivator 4 (NCOA4) is a selective cargo receptor that mediates the autophagic degradation of ferritin under iron-depleted conditions. The protein is predominantly localized in the cytoplasm with 624 amino acids and a MW of about 70 kDa. It contains four crucial domains, an N-terminus coiled-coil domain responsible for protein oligomerization, two conserved motifs characteristic for nuclear receptor interaction (LXXLL and FXXLF), and a ferritin binding site located at the C-terminal domain. The NCAO4 AlphaFold structure prediction showed a very unstructured protein consisting of a unique longitudinal alpha-helix, several short and random alpha-helices, and a few unstructured loops. The protein expresses in good amount but is mostly present in the form of insoluble protein aggregates. Previous efforts to solubilize the protein have largely failed, mainly because of an unstructured N-terminal coiled-coil domain. Here, we present experimental conditions that enabled the refolding and re-solubilization of the full-length NCOA4 protein. Current attempts are underway to obtain its structure by Cryo-EM
Sunday
3741704 - Functional asymmetry and chemical reactivity of CsoR family persulfide sensors | Poster Board #2815
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.
Sunday
3741847 - Studies toward the development of DDAH-tag, a novel self-labeling fusion protein system | Poster Board #2417
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Cellular and molecular biology is concerned with a number of fundamental questions regarding proteins, including their function, their half-life, and their localization within the cell. By determining how these processes differ between wild-type cells and disease models, new therapeutic avenues can be discovered and developed. Intrinsically fluorescent proteins are quite useful in this regard, but great strides have been made to develop self-labeling fusion proteins that are modular and more efficient. Recently, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) was found to be selectively and covalently inhibited by 4-halopyridines via nucleophilic aromatic substitution. It was recognized that this novel covalent interaction could lead to the development of a “DDAH-tag”, a self-labeling protein system that could be complementary and orthogonal to existing approaches like SNAP-tag® and HaloTag. Current results will be presented in the endeavor to repurpose DDAH as a self-labeling fusion protein tag, including preliminary data of a simplified model system. Using a “bump-hole” approach, a number of disubstituted 4-halopyridine probes have been synthesized and assessed against their ability to bind to both wild-type DDAH and mutant DDAH, which has a larger active site pocket due to the loss of a histidine residue. To expedite an assessment of DDAH’s self-labeling potential, DDAH has been fused to bovine serum albumin (BSA) at the protein level via tetrazine ligation. Future work will investigate DDAH fusion at the genetic level and optimization of the best halopyridine “bump” to enhance its kinetic profile. It is hoped the DDAH-tag will be a useful and novel contribution to the ever-expanding toolbox available for biologists.
Sunday
3741891 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3742036 - Synthesis and quantitative analysis of diffusible signaling factors, for determination of structure activity relationships and structural stability | Poster Board #2817
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Biofilms pose a threat to public health. The National Institutes of Health estimate that biofilms are linked to between 65 and 80% of hospital-related infections. Biofilms are a complex network of bacterial cells contained within an extracellular matrix and are 10-1000 times more resistant to common antibiotic treatments. Biofilms utilize a form of intercellular communication known as quorum sensing to regulate many bacterial functions including the excretion of the extracellular matrix and dispersal of planktonic cells. Compounds known as diffusible signaling factors (DSF) regulate the formation/degradation of the extracellular matrix. One family of DSF molecules consists of a carboxylic acid headgroup, a C2-C3 cis alkene, and an alkyl tail. Cis-2-decenoic acid (C2DA) can disperse gram-positive and gram-negative bacteria derived biofilms. However, C2DA is susceptible to oxidative degradation and isomerization, which both lead to a loss of activity. 2-heptylcyclopropyl-1-carboxylic acid (2CP) avoids this degradation by substitution of the cis-alkene with a cyclopropyl group. 2CP is a chemically stabilized form of C2DA that retains antibiofilm activity. We have expanded our analysis of DSF analogs to include alkoxy substituted benzoic acids as mimics of the cis-α/β unsaturation in C2DA. To date a series of 12 alkoxy benzoic analogs have been developed. These analogs differ in position (ortho, meta, para), overall chain length, and chain branching. Additional modification of the carboxylic acid headgroup is also in progress. Antibiofilm activity of these novel diffusible signaling factors will be discussed.
Sunday
3742066 - Self-organization of designer proteins into lipid membranes based on first principles | Poster Board #2819
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Membranes and membrane-proteins possess complementary physiochemical properties which are thought to drive proper protein sorting and function within cells. However, the extent to which biophysical properties, such as hydrophobic thickness, can drive protein sorting on their own remains unknown. Inspired by this question, we used de novo protein design, molecular dynamic simulations, and cell-free systems to elucidate how membrane-protein hydrophobic mismatch affects protein integration and organization in synthetic lipid membranes. We found that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the cell-free expression and co-translational insertion of membrane proteins into synthetic membranes. Membrane-protein hydrophobic mismatch can be leveraged to sort proteins both between and within membranes, enabling specific cargo delivery and controlled split protein assembly. Therefore, lipid-protein interactions, driven through hydrophobic mismatch are sufficient to sort proteins within membranes within in vitro systems, demonstrating the potential of this biophysical interaction to drive protein localization in cells as well as engineer new membrane-based materials.
Sunday
3742080 - Peptide targeted lipid nanoparticles capable of delivering therapeutic payloads selectively into tumor associated macrophages (TAMs) for the activation of anti-cancer effector functions | Poster Board #2620
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Cancer cells maintain intricate interactions with their surrounding stroma within the tumor microenvironment (TME). Tumor associated macrophages (TAMs) are part of tumor stroma that orchestrate various aspects of immunity, such as tumor progression, angiogenesis, metastasis, and tumor relapse. Unfortunately, the lack of effectiveness observed in clinical trials due to the indiscriminate targeting of all macrophages and unwanted depletion of macrophages in other organs, have resulted in weak efficacy and limited clinical translation of existing strategies to treat cancer. While most targeted approaches focus on destroying tumor cells, combating immunosuppressive cells in TME is still in its early stage. In this work, we are developing lipid nanoparticles (LNPs) capable of selectively targeting TAMs to modulate the immunosuppressive effects for efficient therapeutic outcomes. We designed and optimized LNPs containing Prodrug to selectively reprogram TAMs to activate anti-cancer effector functions for efficient cancer treatment. The key aspect of this research is selective therapeutic delivery into specific cells by designing and functionalizing LNPs using peptide-targeted approach to overcome non-specific delivery.
Sunday
3742217 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3742234 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3742278 - FMOC Leucine amino acid inhibitors exemplify strong noncovalent interactions in complex with butyrylcholinesterase enzyme | Poster Board #2528
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The formation of beta amyloid plaques is a prominent indicator of the progression of Alzheimer’s Disease (AD); this symptom is also associated with overactivity of the butyrylcholinesterase (BChE) enzyme, which hydrolyzes the neurotransmitter acetylcholine, making it a plausible target for the treatment of this neurodegenerative disease. Here, computational methods were used to probe the interactions between the enzyme and inhibitor candidates featuring 9-fluorenylmethyloxycarbonyl (Fmoc). Using Molsoft ICM-Pro software, 10000 docking trials of 12 Fmoc inhibitors were performed, and trials resulting in the most optimal docking scores were examined. The best candidate was Fmoc-Norleucine, consistent with experimental IC50 values. Analysis of the best-scoring structures show that the length of the aliphatic R group in inhibitors was most influential on resulting docking scores, as the four inhibitors with the largest side chains (L-Norleucine, D-Norleucine, L-Homoleucine, and D-Homoleucine) had the most favorable docking scores. From visual analyses of the enzyme in complex with these inhibitors, their larger aliphatic groups allowed for more significant ��-stacking between Fmoc and peripheral aromatic site residues of BChE, as well as orientation of the terminal carboxylate of inhibitors farther into the enzyme active site near the oxyanion hole and catalytic triad, both of which are hydrogen-rich and thus favor hydrogen bonding interactions. Identification of trends observed in experimental and in silico studies provides a better foundation for future design of selective amino acid-based BchE inhibitors.
Fmoc group of L-Norleucine situated in left of BChE binding pocket, near catalytic triad aromatic group, indicative of π-stacking interactions occurring.

Fmoc group of L-Norleucine situated in left of BChE binding pocket, near catalytic triad aromatic group, indicative of π-stacking interactions occurring.


Sunday
3742583 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3742592 - Metabolic and morphological perturbations associated with developed resistance to metal oxide nanomaterials in Shewanella oneidensis | Poster Board #2429
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Lithiated nickel manganese cobalt oxide (NMC) nanomaterials are currently used as a battery cathode in electric vehicles due to their high energy storage capacity and low cost. The soring production rates of electric vehicles in conjunction with improper nanomaterial disposal protocols has the potential to result in greater concentrations of NMC in the environment. Previous work has shown that acute exposure of NMC to Shewanella oneidensis, a ubiquitous gram-negative environmental bacterium, is toxic; yet, chronic NMC exposure elicits bacterial resistance. Current work is centered around elucidating the mechanism by which S. oneidensis becomes resistant to NMC. To identify specific cellular components implicated in NMC resistance, we compared the response of resistant and wild-type bacteria to a variety of stimuli (nutrients, osmotic pressure, etc.). We found that a nutrient-rich environment induces a unique change in the morphology of NMC-resistant cells. This unusual response to nutrients implies that NMC-resistance perturbs cellular metabolism. This initial understanding of acquired NMC resistance in S. oneidensis not only illuminates the broader ecological consequences associated with introducing nanomaterials into the environment, but also provides mechanistic insight into the larger tapestry of bacterial resistance.
Sunday
3742728 - Validation studies of novel molecules targeting bacterial aminodeoxychorismate synthase | Poster Board #2435
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
According to the CDC, antimicrobial resistance is responsible for more than 2.8 million infections each year in the United States, with at least 35,000 deaths attributed to resistant infections. To address this public health issue, the development of new antimicrobial agents targeting validated antibacterial pathways are needed. Recently, our lab became interested in folate biosynthesis. This pathway is a validated target where existing drugs such as sulfamethoxazole and trimethoprim target dihydropteroate synthase and dihydrofolate reductase, respectively. Part of the pathway that has received limited attention is the synthesis of the crucial folate precursor, para-aminobenzoic acid (PABA). PABA is produced from chorismate by the action of the glutamine-dependent aminodeoxychorismate synthase (ADCS) and aminodeoxychorismate lyase (ADCL). Previous work has shown that inhibition of ADCS is antibacterial, yet known inhibitors are riddled with issues such as the rapid development of resistance and mammalian cell cytotoxicity. It is notable that there have not been any high-throughput screening campaigns published that have targeted ADCS. To overcome this barrier, we are presenting the results of a high-throughput screening campaign conducted using a thermal binding assay. Hits identified after primary screening were subject to follow-up dose dependent studies including validation in an absorbance assay. Our findings will be presented in this poster.
Sunday
3742730 - Popping the lid off: Rare bicyclic sesquiterpene may emerge from a tricyclic intermediate | Poster Board #2433
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Sesquiterpene cyclases catalyze the conversion of farnesyl diphosphate, a linear compound, into a dizzying array of molecular structures including monocyclic, bicyclic, tricyclic, and even tetracyclic products. These products include both sesquiterpene hydrocarbons and sesquiterpenoids containing heteroatoms (typically oxygen). The details of the chemical mechanisms catalyzed by these enzymes, including stereochemical requirements, are preserved in the molecular structures of their products. Using computational chemistry methods, models for the chemical mechanisms by which these enzymes convert farnesyl diphosphate into a range of products can be elucidated. Patchoulol synthase isolated from Pogostemon cablin (patchouli), the natural source of patchouli oil (a popular fragrance), converts farnesyl diphosphate into patchouli alcohol (a tricyclic sesquiterpenoid) plus a range of bi-, tri-, and tetra-cyclic sesquiterpene hydrocarbons. Among these products is δ-guaiene, a rare sesquiterpene that contains a bicyclo[5.3.0]decane framework improbably derived directly from transannular ring closure of a putative ten-membered ring intermediate. Rather, the mechanism proposed herein suggests the bicyclo[5.3.0]decane framework leading to delta-guaiene may emerge from "popping the lid off” of a tricyclic intermediate identified along the pathway leading toward patchouli alcohol. In summary, this mechanism explains the generation of patchouli oil as well as δ-guaiene and other products of this enzyme with stereochemical precision. Intriguingly, this mechanism also implies the nature of the evolutionary relationship between the well-characterized patchoulol synthase from Pogostemon cablin and the less-well characterized δ-guaiene synthase isoforms isolated from Aquilaria crassna (agarwood).
Sunday
3742769 - Discovery of C13-aminobenzoyl cycloheximide derivatives as potent inhibitors of translation elongation | Poster Board #2530
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Yumi Koga, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Employed for over half a century to study protein synthesis and commonly used in ribosome profiling to map global ribosome positions, cycloheximide (CHX) is a small molecule natural product that reversibly inhibits translation elongation. Despite its extensive use, CHX treatment often results in incomplete translation inhibition due to its limited potency and rapid reversibility. Here we report the concise and scalable synthesis of C13-amide functionalized CHX derivatives with increased potencies toward translation elongation inhibition. Structure determination by cryogenic electron microscopy revealed that C13-aminobenzoyl CHX derivative occupies the same site as CHX to compete with the 3' end of E-site tRNA. We demonstrate that the C13-aminobenzoyl CHX derivative is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.
Sunday
3742823 - Investigation of structural factors controlling loop dynamics in acyl protein thioesterases | Poster Board #2437
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Acyl protein thioesterases catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Recently, human APTs were shown to use a two-step catalytic mechanism involving initial membrane binding of an exposed flexible loop followed by substrate binding and cleavage. This two-step catalytic mechanism is likely conserved as a bacterial homologue of human APTs is also regulated by the large-scale movement of an essential dynamic loop upon membrane binding. However, the causes for the movement of this dynamic loop in APTs are unknown. Herein, we investigated factors that trigger loop movement in human and bacterial APTs. In particular, APTs were assayed with inhibitors structurally similar to long chain lipids and to membrane-mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane-mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.
Sunday
3742866 - Interactome mapping by photoaffinity labeling of proteasomal activator and FDA-approved drug Miconazole | Poster Board #2500
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease, share a common feature: aggregation of intracellular proteins. In eukaryotic cells, the proteasome system is responsible for most of the non-lysosomal protein clearance, and it can degrade proteins regardless of their ubiquitinated state. As cells age, the proteasomal activity declines due to a decreased subunit expression or disassembly. This proteostasis malfunction leads to protein aggregation and, subsequently, diseased states; hence, the discovery and evaluation of small molecules that can restore proteasomal activity levels, and clear aggregation-prone proteins, represents an attractive therapeutic approach. Advances in the field have identified molecules that increase the proteasome-mediated proteolysis; however, no studies have yet accurately identified their binding site and addressed their off-target toxicity. Recently, we identified the FDA-approved drug miconazole (MO) as a stimulator of the 20S proteasome isoform and validated its activity profile in biochemical and cell-based assays. Given its FDA-approved drug status, we considered that to successfully repurpose it, information regarding its mechanisms of action and interaction network at a proteome-wide scale is necessary. Here, we’ve used a photoaffinity labeling (PAL) approach to map MO’s interaction network. Through structure-activity relationship (SAR) studies, we’ve determined MO’s key features responsible for proteasomal activation and designed a diazirine-based photoreactive probe that covalently binds to MO’s binding partners in cellular environments. Currently, we are working towards an affinity enrichment step followed by tandem mass spectrometry of photo cross-linked targets to identify the biological targets, and we will subsequently validate them by biophysical or genetic methods.
Sunday
3742972 - Optimization of in vitro inflammatory caspase assays using peptide and small molecule activity-based probes | Poster Board #2502
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Inflammatory caspases (caspase-1, -4, and -5 in humans) are key players in the innate immune response, although the individual role of each enzyme is not well understood. All three enzymes have been shown to cleave essential pro-inflammatory substrates, such as Gasdermin D, implicating them in many inflammatory disease states. The optimal conditions for activation of each enzyme appear to be distinct and require further investigation to determine whether their roles are interconnected or independent. Caspase-1 is the most active and the best understood family member. It is thought to be a dimer in its active form, while caspase-4 and -5 have been reported to be dimers or high molecular weight oligomers when active. Studying the activation is further complicated by differing degrees of autoproteolysis for caspase-1, -4, and -5. To interrogate the roles of these enzymes, new tools are needed to uncover more detailed information regarding the activation conditions and structural state of each of the inflammatory caspases. We have used a combination of small molecule and peptide-based probes to examine activity and inhibition profiles for each of the caspases. We will present the optimal conditions we have determined for each enzyme, as well as the kinetic data for inhibitors and substrates.
Sunday
3742985 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743085 - PUFA phospholipid bilayer structure determination using neutron and X-ray scattering data | Poster Board #2900
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Polyunsaturated fatty acids (PUFAs), commonly known as omega-3 and omega-6 fatty acids, have been shown to prevent chronic illnesses when consumed; PUFA-containing lipids are critical to organism development and membrane function. Currently, research on the structure of PUFA phospholipids is in its nascent stages, leaving much room for further characterization of this group of lipids. Through a combination of small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations, the structures of five PUFA phospholipids were determined, namely: 1,2-diarachidonyl-phosphatidycholine (DAPC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (DDPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC), and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC). Structural parameters including area per lipid, total bilayer thickness (DHH), hydrocarbon thickness (2DC), and the area compressibility modulus (KA) were analyzed from scattering density profiles (SDPs) determined from fits to the scattering data, as well as unconstrained and area-constrained MD simulations using the C36 CHARMM force field. Areas per lipid determined from the SDPs and (unconstrained) MD simulations at 20oC and 30oC (except for DDPC) were found to be in good agreement with one another. However, when analyzing DHH and 2DC, the resulting experimental SDP values were approximately 10-15% lower than the values obtained from MD simulations. Area-constrained simulations allowed for determination of the area compressibility modulus and the area per lipid across simulated and scattering data. Joint analysis of SANS and SAXS data along with MD simulations provides an accurate protocol for bilayer structure of PUFA phospholipids and the use of these different, but complementary, techniques offers insights into the discrepancies that are observed within this unique class of biologically relevant lipids.
Sunday
3743131 - Design of novel benzothiazole carboxamide based HK2/EGFR- dual kinase inhibitors: A green chemistry approach | Poster Board #2902
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Epidermal growth factor receptor (EGFR) and Hexokinase 2 (HK2) overexpression are associated with the progression of oral cancer. However, none of the small molecule EGFR or HK2 inhibitors are available clinically for the treatment of oral or oropharyngeal cancers. Thus, a series of benzothiazole carboxamide based EGFR and HK2 dual kinase inhibitors are designed following structure-guided drug design strategies with the idea of drug repurposing. One representative compound (PC-BTZ-L) from our library exhibited better growth inhibitory properties in FaDu and Cal27, oropharyngeal and tongue cancer cell lines respectively. The dual kinase inhibitor also induced apoptosis and exhibited cell cycle arrest of FaDu cells at a concentration of 30 µM. Further cellular assays revealed that PC-BTZ-L antagonises glucose uptake 72 hours post treatment as indicated by the reduced mean fluorescence intensity of 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose) in FaDu cell line. Treatment of FaDu cells with 30 µM of PC-BTZ-L for 72 hours also showed inhibition of mitochondrial membrane potential as indicated by reduced mean fluorescence intensity of tetra methyl rhodamine ethyl ester (TMRE) suggesting mitochondrial toxicity of the kinase inhibitor. Moreover, the newly established synthetic strategy of PC-BTZ-L and other compounds in the series involved direct amidation of esters by chelation of cation of the used base and the final compound was separated without performing any column chromatography reducing the time and cost of the synthesis procedure.
Fig.: Green synthetic strategy and <i>in vitro </i>studies of benzothiazole carboxamide based HK2/EGFR- dual kinase inhibitor.

Fig.: Green synthetic strategy and in vitro studies of benzothiazole carboxamide based HK2/EGFR- dual kinase inhibitor.


Sunday
3743137 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3743141 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743257 - Deciphering the role of inflammatory ques on cancer progression in the tissue microenvironment | Poster Board #2506
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
High mortality rates of many cancer types can often be attributed to instances where metastasis occurs before detection and recurrence after treatment. These phenomena are often associated with the persistence of cancer stem cells (CSCs), a small population of cells with an increased chemoresistance and tumor-initiating capacity, after initial surgical debulking. Some CSCs can be identified through surface biomarkers (e.g., CD133), but often they are more reliably isolated based on an increase in aldehyde dehydrogenase (ALDH) activity, which is widely considered a universal CSC marker. ALDHs are enzymes that catalyze the oxidation of aldehydes to carboxylic acids, which makes these enzymes crucial for drug detoxification and stemness signaling (via retinoic acid metabolism). While ALDHs provide crucial function to CSCs, the regulation of these enzymes is relatively unknown. Therefore, a crucial first step in understanding CSCs is to investigate what factors are responsible for the regulation of ALDHs. In recent years, the tumor microenvironment has been shown to be a crucial mediator of cancer progression, therefore in this work we have focused our efforts on determining how the tumor microenvironment may affect ALDH activity. To this end we have employed a variety of techniques ranging from fluorogenic probes, small-molecule inhibitors, cell and tissue models, molecular biology, and proteomic analysis. Our results shed new light in how crosstalk at the molecular level can influence cancer progression via ALDH modulation.
Sunday
3743263 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743269 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743305 - Exploring viperin’s role in the degradation of viral proteins using a membrane mimetic system | Poster Board #2713
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Viperin is a conserved ER membrane-associated protein encoded by an interferon-stimulated gene and has demonstrated antiviral activity. While viperin’s antiviral activity can be attributed to its radical SAM-dependent synthesis of the chain-terminating nucleotide ddhCTP, which interferes with viral replication, it has also been shown to interact with various cellular pathways implicated in viral infection. Furthermore, viperin interacts with viral proteins that localize to the endoplasmic reticulum, such as NS3, in Tick-Borne Encephalitis and Zika virus infections, and NS5A in Hepatitis C virus infections. Experiments using proteasomal inhibitors suggest that viperin stimulates the proteasomal degradation of both viral proteins, though the exact mechanism is unknown. Viperin has not been shown to possess a ubiquitin binding domain and is thought to interact with an E3 ubiquitin ligase while complexed with viral protein. Furthermore, replicating these interactions in vitro using constructs lacking the membrane-binding domain has proved challenging. Viperin has recently been incorporated into nano discs by our group, and we present recent progress aimed towards optimizing and characterizing this system. This research will facilitate in vitro studies of the enzyme interacting with other ER-membrane associated proteins by providing a native-mimetic environment. Specifically, the development of a nano disc system incorporating viperin will allow us to characterize complex formation of viperin and viral proteins. Furthermore, this system will be applied to probe the interactions of E3 ligases that, mediated by viperin, lead to the proteasomal degradation of viral protein. This will further help establish an additional mode of action for viperin’s antiviral activity. Ultimately, understanding how viperin may act as an agonist of proteasomal degradation would elucidate its roles both in antiviral defense and cellular metabolic regulation.
Sunday
3743382 - Inhibition of a major metabolic pathway in prostate cancer by a cisplatin prodrug | Poster Board #2716
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The dependency of a large population of tumors on fatty acid oxidation (FAO) as one of their main carbon sources in very common in the field of cancer metabolism. This is one of the irregularities that tumorigenic cells portray in their metabolic profile. The shift to mitochondrial oxidative phosphorylation (OXPHOS) from glucose oxidation or glycolysis despite large availability of oxygen has been known now for quite a long time but developing new targeting strategies that can inhibit this transition is need of the hour. Fatty acid synthase inhibitors have been reported to show profound effect on malignancies specially cancers of mammary glands, prostate, and gliomas. Cisplatin, oxaliplatin, carboplatin etc., the FDA approved platinum anticancer drugs are used against a vast variety of cancers. Prostate cancer (PCa) is such a type of cancer which is resistant to cisplatin. The metabolism in such type of malignancy depends heavily on the energy derived from fatty acid oxidation (FAO). FASN is involved in catalyzing the synthesis of long-chain saturated fatty acids and is overexpressed in many cancer types. FAO provides the energy required for metabolic needs even after glycogen depletion which results in elevated glycolysis. Various studies have also revealed that carnitine palmitoyl transferase (CPT-1A), a mitochondrial protein is the rate limiting protein for mitochondrial β-oxidation, is elevated in PCa cells. It has also been observed that patients who are treated with androgen deprivation therapies has high expression of certain proteins which are dependent on androgen and drive the metastatic potential of PCa and ultimately lead to castrate resistant prostate cancer (CRPC). Here we propose that a cisplatin prodrug, unlike other conventional platinum drugs or cisplatin prodrugs causes inhibition in one of the major metabolic pathway that the PCa cells depend upon. Our work with Platin-L will show us the exact mechanism of how the are dependent on fatty acids over glucose and glutamine and how the drug alters their lipogenesis profile. The inhibition of FAO in tumor cells will make them more vulnerable and apoptotic. We are on our way to find the suitable conditions which should suppress the mitochondrial FAO and make the PCa cells sensitive towards cisplatin-based chemotherapy. We also propose a suitable vehicle for the delivery of our prodrug to the tumor cells in the form of orally administered nanoparticles and specifically target PCa.
Sunday
3743463 - Development of a non-perturbing photoacoustic formaldehyde sensor | Poster Board #2508
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Several fluorescent probes have been used to study reactive formaldehyde (FA) in biological systems. However, many of these probes use an activation mechanism that depletes FA from the biological system it is investigating, so an accurate concentration for FA cannot be determined. While a probe has been developed that regenerates any FA that is detected, it relies on a fluorescence read-out, which is poorly suited for in vivo studies due to poor image resolution and low signal. To counteract these issues, we have developed a FA-regenerative photoacoustic (PA) probe. PA probes use a “light in, sound out” approach to achieve high-resolution images at an imaging depth in the centimeter range, which make them an attractive alternative to fluorescent probes. Our design for the FA-regenerative probe consists of a PA dye, a FA-reactive trigger, and a masked FA-regenerating moiety. After the FA reacts with the trigger, the FA-regenerative moiety unmasks to form the unstable hemiacetal, which immediately decomposes to release the free PA dye. We hypothesize that after the probe reacts with formaldehyde, the chemical change in its structure will enable us to selectively visualize the reacted and unreacted probes, which will enable us to quantify the amount of FA present and carry out ratiometric imaging. We plan to use this technology to accurately monitor FA levels in the brain in relation to DNA methylation and memory, which has previously unable to be done without depleting endogenous FA.
Sunday
3743481 - Fine-tuned small molecule fluorophores for fluorescence-guided surgery | Poster Board #2702
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
As many as 40 million surgeries are performed annually in the United States for both acute and chronic interventions, with a large portion performed without any image guidance, forcing surgeons to rely solely on direct visualization and palpation. In the context of tumor resection, this results in poor margins that lead to high rates of recurrence, and is confounded by iatrogenic nerve injury as a result of poor visualization and tissue penetration from white light alone. Fluorescence guided surgery (FGS) has shown significant promise in combatting these shortcomings of typical surgery through the use of a wide range of fluorescent markers, from antibodies to small molecules. Compared to bio-affinity agents and nanomaterials, small molecules are more desired for in vivo fluorescence imaging, owing to their low toxicity, low production cost, quick tissue uptake, and rapid clearance. Previous work has identified the oxazine scaffold as a promising avenue for nerve-specific contrast agent development, due to its sufficiently low molecular weight to cross the blood-nerve-barrier (BNB), tunable photophysical properties, and high nerve specificity, all of which underly the importance of elucidating structure-function relationships through functional modification. The two main strategies behind the optimization of small molecules for nerve-sparing FGS involve modifications to increase nerve specific uptake, as compared with muscle and adipose tissue, as well as photophysical tuning for near-infrared (NIR) absorption and emission profiles, signal intensity, and penetration depth, among others. The clinical utility of these novel small molecule fluorophores will be demonstrated through mutlti-color FGS, enabling simultaneous visualization of critical nerve and tumor tissues.

Sunday
3743554 - Structural and dynamical responses to small-molecule inhibitors targeting a new allosteric site in PTP1B | Poster Board #2810
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Protein Tyrosine Phosphatase 1B (PTP1B) is the archetypal member of the PTP family and a validated target for a number of diseases including diabetes and cancer. The conserved active sites of PTPs are highly polar and present challenges for the development of specific and bioavailable small-molecule modulators. Allosteric sites on proteins have long been understood to be of potential use in the development of specific targeted therapeutics, but the mechanisms that convey a signal through a protein’s structure remain poorly understood. A recently identified L16 allosteric site in PTP1B is of particular interest because of its lack of conservation amongst the PTPs. To explore the allosteric potential of the L16 site, we have used biophysical experiments to characterize the structural and functional effects of new compounds that were designed to target this site using the Atomwise AtomNet artificial intelligence system applied to crystal structures from a previous fragment soaking screen of PTP1B. Among the top predicted compounds, we have identified both high-affinity but benign binders, and inhibitors. The structural and dynamical changes these compounds brought about in PTP1B were further analyzed using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and X-ray crystallography. Despite being targeted to the same L16 binding pocket, these ligands induce distinct structural and dynamical responses in PTP1B, suggesting that the two inhibitors may involve different loci in PTP1B’s structure in conveying their modulation. Overall, this work provides promising new allosteric footholds for the long-studied therapeutic target PTP1B.

Sunday
3743593 - Nanomaterial-modified electrodes for improving the performance of microbial fuel cell | Poster Board #2515
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Microbial fuel cells (MFCs) which convert the chemical energy into electrical energy through bacterial metabolism have emerged as an alternative energy source. Although there is a great potential for MFCs application, it still faces practical barriers such as low voltage output and current density. The development of eco-friendly electrodes, which can improve the electron transfer capacity is crucial to improve the MFC performance. Here, to facilitate the interactions between electron generating bacteria and electroactive surfaces in anode, polyaniline (PANI) was electropolymerized on the carbon felt (CF) surface followed by the gold nanoparticles (AuNPs) electrodeposition. The PANI helps in the attachment of bacterial cells, while the subsequent AuNPs deposition increases the effective surface area and electrical conductivity of the resulting electrode. Moreover, DNA/chitosan nanocomposite is used as a new type of cathode material, which is fabricated by physiochemical adsorption of rectangular DNA origami nanostructure and chitosan onto CF. The DNA/chitosan composite acts as electron acceptor, which eliminates the frequent replacement of oxidizer in cathodic chamber intensifying the MFC sustainability. The modification of CF cathode with chitosan and DNA nanostructure was observed by CV and electrochemical impendence spectroscopy. The performance of MFCs with AuNPs/PANI modified anode and DNA nanostructure/chitosan fabricated cathode were compared and found more effective than non-modified electrodes with higher both current density, and voltage output capability. Our results provide a simple and controllable method for the preparation of high-performance and cost-effective MFC.
Sunday
3743790 - Role of a four-helix bundle protein's loop in its stability and activity | Poster Board #2532
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
While we know a lot about the role of hydrophobic core packing in protein folding and stability, we still need more information about the role of other parts of a protein in its folding, stability, and activity. We have used a homodimer four-helix bundle protein (Rop) to investigate the role of its turn in the stability and activity of the protein. Rop’s tight turn has four amino acids (L29 D30 A31 D32) that connect two helices of a monomer. We made four libraries comprising all positions in the turn and selected active variants with a cell-based screen. Position 29 showed mostly medium-sized hydrophobic residues, and D30G showed up frequently in position 30, which is a known stabilizing mutation. For position 31, the most abundant amino acid was surprisingly tryptophan. Also, although we hypothesized that a surface exposed ionic bond between D32 in the loop of a monomer and R55 of the other monomer may cause a bias in position 32 toward negatively charged amino acids, our libraries showed little bias in this position. In addition to measuring the thermal stability of selected library variants, we also rationally designed some variants to study the role of individual mutations in the overall stability and activity of the protein. We found although W is rarely seen in turns, A31W stabilizes Rop significantly. We also found that while most 5 amino acid turns in the library were somewhat destabilized, others such as one with a single Gly insertion are extremely stable. Rop’s turn tolerates a wide variety of amino acids, but it can play an important role in the stability of the protein. These results shed light on competing factors in turn and loop stability generally, as well as revealing some surprising routes to stabilization in Rop in particular.
Sunday
3743819 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743839 - Design and evaluation of NIR-I and -II dyes with enhanced solubility and targeting | Poster Board #2514
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The use of near-infrared (NIR) imaging agents is a promising avenue for the diagnosis and treatment of cancer. NIR dyes are advantageous since endogenous pigments (e.g., hemoglobin, deoxyhemoglobin, and melanin) do not absorb substantially in the NIR window, allowing light to reach deeper regions of the body. Two promising modalities that have emerged for these purposes are photoacoustic (PA) and fluorescence imaging. Several different dyes have been optimized for NIR-I (650 to 900 nm) and NIR-II (900 to 1700 nm) PA and fluorescence imaging (e.g. aza-BODIPY, cyanine, xanthenes, etc.). However, in order to absorb/emit in the NIR-I and -II regions, these dyes are large, hydrophobic, and prone to aggregation which can negatively affect solubility and tumor targeting. To address these shortcomings, we strategically installed modifications to the aza-BODIPY and cyanine platforms to enable enhanced solubility and cancer targeting. These modified dyes were evaluated in the in vitro and in vivo setting.
Sunday
3743904 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3743972 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3744235 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3744398 - A general method for chemogenetic control of peptide function | Poster Board #2526
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Natural or engineered peptides serve important biological functions. A general approach to achieve chemical-dependent activation of short peptides will be valuable for spatial and temporal control of cellular processes. Here we present a pair of chemically-activated protein domains (CAPs) for controlling the accessibility of both the N- and C-termini of a peptide. CAPs were developed through directed evolution of an FK506 binding protein (FKBP). By fusing peptides to one or both CAPs, their function is blocked until a small molecule displaces them from the FKBP ligand binding site. We demonstrate that CAPs are generally applicable to a range of short peptides, including a protease cleavage site, the SsrA peptide which dimerizes with the SspB protein, and a nuclear localization signal peptide, with a shield-1 dependent dynamic range up to 156-fold. We also show that the CAPs system can be utilized not only in mammalian cell culture, but also in multiple organs in living animals. Therefore, we have established a novel chemogenetic approach to control peptide activity in vitro and in vivo.
Sunday
3744468 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3744651 - Design of protein lock copolymers for water-responsive actuation | Poster Board #2908
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Natural water-responsive (WR) biological materials are abundant, having independently evolved in places such as wheat awns, pinecones, and bacterial spores. Water-responsive biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural fluctuations in humidity. Recent work on water responsive protein materials has shown that β-sheet structure correlates with WR energy density, but the design parameters for water response in proteins remain poorly understood. Here we design, synthesize, and study protein block-copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E). We use these protein materials to create water-responsive actuators whose energy densities outperform mammalian muscle by two orders of magnitude and match reported spider silk actuators, the highest found in nature. To elucidate the effect of structure on water response, CEC was compared to a variant, CECL44A, in which the Leu at position 44 in each C domain was mutated to Ala, abolishing any α-helical structure and providing an unstructured control material. In this work, we study the relationship between protein secondary structure, higher-order protein assemblies, and WR energy density in order to develop design parameters for water-responsive biomaterials.
Sunday
3744759 - Biosynthesis and characterization of bioplastics using a native bacterial strain and cacao fruit residues | Poster Board #2910
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Increasing material's circularity in agroindustrial crops is of fundamental importance for a low-carbon economy. We are interested in improving Colombia's cacao production circularity by increasing biomass usage. Processing the whole cacao fruit is a strategy that allows access to residual biomass, such as cacao mucilage exudate (CME), to produce advanced materials. Liquid cacao fruit wastes (CLWs) result from biotechnological applications of CME. For instance, spent cacao mucilage exudate (SCME) originates from cacao beans fermentation. At the same time, from biocellulose synthesis, we obtain residual media from bacterial cellulose production (RMBC). SCME and RMBC are effluents rich in sugars and low molecular weight organic acids.
In this work, we report using CLW to formulate various culture media for polyhydroxyalkanoates (PHAs) biosynthesis using a native Bacillus megaterium strain (B2). B2 accumulates intracellular PHA inclusions as carbon and energy reserves. We follow the adaptation process of B2 to the CLW-based media and the microorganism development in terms of acids and sugars consumption. Once adapted, the strain was transferred to a 4-liter batch bioreactor to assess the influence of operational variables in PHAs production, yield, productivity, and microorganism kinetics. The product yield was 0.98 g / g, with an accumulation percentage of 57% and a productivity increase of 33% compared to other works using the same microorganism. Characterization using MALDI-TOF-MS, FTIR and UV-vis spectroscopy, elemental composition, and thermal analysis confirmed the presence of a bioplastic of the polyhydroxybutyrate (PHB) type.

Sunday
3745179 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3745198 - Extraction, preservation, and concentration of SARS-CoV-2 viral RNA from saliva and gargle | Poster Board #2918
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Control, containment, and mitigation of the coronavirus disease 2019 (COVID-19) pandemic require accessible tools and assays for the timely detection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from patient samples. Because saliva and gargle samples are non-invasive, easy to collect, and abundant, they emerge as favorable alternative sampling options to the conventional nasopharyngeal swab (NPS). However, saliva and gargle are more complicated, and viral particles are often present at lower levels. The objective of this research is to develop a rapid, efficient, and simple method for extracting, preserving, and concentrating SARS-CoV-2 viral RNA from saliva and gargle samples.
The SARS-CoV-2 viral RNA contains a 3' polyadenylated tail similar to messenger RNA. By taking advantage of this unique 33-residue poly-A tail, we developed a rapid, efficient, and simple viral RNA extraction method using poly-T conjugated magnetic beads. We also developed an extraction buffer enabling efficient lysis of viral particles and preservation of the released viral RNA in saliva and gargle samples. The released viral RNA remains stable at ambient temperature for more than 2-weeks in our buffer. The viral RNA extraction can be completed within 15 minutes under room temperature.
Using this extraction method, we achieved near 100% recovery of SARS-CoV-2 viral RNA and were able to detect as few as 10 viral particles in 100 µL of saliva or gargle samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR). We successfully coupled this method to a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for point-of-care detection of SARS-CoV-2 in saliva and gargle.

Sunday
3745346 - Fluorescent cationic conjugated agents for effective antibacterial application | Poster Board #2534
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Because of the increasing antibiotic resistance of the pathogens, new materials with significant antibacterial effect are highly demand. In this work, a series of fluorescent cationic conjugated agents are designed to interact with two different bacteria, Gram-negative E. coli and Gram-positive S. aureus. The reactive oxygen species (ROS) of these cationic conjugated agents are generated under light irradiation, which can effectively kill the bacteria. Meanwhile, the fluorescent images of the bacteria after incubation and irradiation with different time and concentration of different cationic conjugated agents are recorded using confocal fluorescent microscopy (CFM). With this method, the interaction between bacteria and cationic conjugated agents in real time is monitored, the differences can be compared and further studied.

Sunday
3745535 - Kinetic studies of pyran ring stabilization variants of Campylobacter jejuni NapA | Poster Board #2503
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Periplasmic nitrate reductase (Nap) is a Moco-containing member of the DMSO reductase family. Moco enzymes generally catalyze reactions where there is a net difference of an oxygen atom between the substrate and the product, in this case, the reduction of nitrate (NO3-) to nitrite (NO2-). The pterins bound to NapA Moco can exist in a variety of oxidation states such as ring closed (dihydro) or ring open (fully oxidized). The P-pterin of NapA in the tetrahydro state is involved in electron transfer from NapB. The pterin oriented toward the interior of the protein in the dihydro state, D-pterin, is involved in tuning redox potential.The pyrazine nitrogen atoms of P- and D-pterins differ in terms of their H-bond donor or acceptor ability. These H-bonding networks stabilize the closed ring redox state of the P-pterin preventing oxidative ring opening. Residues W890 and F914 of C. jejuni NapA are located on either side of the P-pterin stabilizing it through a -stacking interaction. Mutations of these amino acids to an alanine are hypothesized to affect -stacking and permit an open pyran ring conformation. Additionally, mutations of H822 and R816 to acidic, negatively charged residues will disrupt the H-bonding network and possibly destabilize the closed ring state of the P-pterin. Both H822 and R816 are H-bond donors, and these mutations may disrupt the H-bond network and slow electron transfer to the active site. The kinetic consequences of these mutations will be discussed in this poster.
Sunday
3745594 - Spectroscopic studies on the interaction of DNA with Ru(II) complexes | Poster Board #2501
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
A series of mononuclear ruthenium(II) complexes have been prepared and their binding affinities toward double stranded native DNA was investigated by means of absorption spectroscopy, polarized light spectroscopy including circular and linear dichroism (CD and LD), fluorescence quenching and DNA thermal denaturation techniques. The complexes produce LD signal consisting of positive and negative signal in the absorption region upon binding to DNA although these complexes exhibited red-shift and hypochromism in the absorption spectrum. These contrasting observations indicated that the binding modes of the complexes are largely deviated from classical intercalative binding. These results suggest that the binding nature of the complexes to DNA is liable over an intercalative mode of binding to DNA. The small increase of DNA melting temperature in the presence of the complex indicates predominantly DNA groove binding propensity of the complexes. Absence of "molecular light switch effect" indeed further supports non-intercalative binding mode with DNA for the new complexes. This conclusion is also supported by the comparison of the resulting data with the [Ru(phen)3]2+ . This study demonstrated the importance of the structure of bridging moiety in the intercalation of the Ru(II) complex.
Sunday
3746247 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3746282 - High throughput screen for novel small molecule inducers of trained immunity | Poster Board #2505
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Recent work has revealed that innate immune cells have a nonspecific form of memory, referred to as “trained immunity” (TI). Trained immunity is characterized by epigenetic and metabolic remodeling that result in improved effector responses to insult. Training the innate immune system improves organism survival against lethal infections or cancer challenges. Current models of trained immunity focus on macrophages and use β-glucan or Bacillus Calmette–Guérin (BCG) vaccines to induce training, both of which are limited in their therapeutic applications. Small molecules may exhibit improved pharmacokinetic and safety profiles over traditional inducers of training. Furthermore, novel small molecule inducers may trigger trained immunity through previously unknown pathways that result in similar epigenetic and metabolic effects, the identification of which could give us greater understanding and control over trained immunity. We use high throughput screening to identify small-molecule inducers of trained immunity in murine bone-marrow derived macrophages (BMDMs). The central hypothesis of this work is that using high throughput screening to identify novel inducers of trained immunity will improve both our ability to train innate immune cells in therapeutic settings and our understanding of various training pathways.
Sunday
3746851 - Role of IGF-2 in the formation and invasion of trophoblast tumors: A functional and transcriptional study of throphoblastic 3D cell cultures. | Poster Board #2628
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
IGF-2 is an important growth factor that promotes proliferation, migration and invasion in trophoblast cells as well as in tumor cells. However, most studies have been performed in mono-layer cell cultures; yet, cancer cell spheroids have been shown to be more physiologically relevant to native tumor tissue than 2D cell culture. While tumor formation requires cell aggregation and the production of extracellular matrix, once formed, oxygen and nutrients gradients arise along the layers and a hypoxic core is formed, which develop different phenotypes inside the tumor. Given these traits of the tumor microenvironment, growth factors might play a different role when used in cells under stress induced by hypoxia and the lack of nutrients which more closely resembles the in-vivo condition. Hence, 3D cell cultures facilitate the study of the effect of growth factors like IGF-2 in tumor or tumor-like cells as the trophoblast in a tumoral microenvironment.

Using an optimized low-cost agarose-based technique for HTR-8/SVneo spheroid formation, IGF-2 was found to promote cell aggregation and it was found that tumor size is regulated by nutrient availability. Conversely, once formed, IGF-2 inhibits the invasion of spheroids in an extracellular matrix when compared with untreated spheroids. Using RT-qPCR, spheroids were found to overexpress some pluripotency genes like OCT4 and NANOG, as well as some malignancy makers as Survivin, HIF-1α and RhoA. However, when stimulated with IGF-2 these markers tended to be expressed differently from 2D cell cultures. These results exhibit that while IGF-2 promotes spheroid formation, it does not promote invasion and it stabilizes the tumor, which points to the potential importance of using spheroids as in vitro model to consider the role of the tumor’s microenvironment in future research.

Sunday
3747517 - Mapping the miRNA regulation of α-1,2 fucosylation | Poster Board #2507
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
MicroRNAs (miRNAs) are short segments of non-coding RNAs that regulate the translation of messenger RNA (mRNA) into protein. Glycogenes, i.e., the genes that control glycosylation, are highly regulated by miRNAs. The glycan motif a-1,2 fucose is an important component of Lewisb and Lewisy and is the defining feature of the blood group H antigen. H antigen is the precursor of the ABO blood group antigens, if it is not present ABO blood group antigens cannot be formed.
FUT1 and FUT2 genes encode a Golgi stack enzyme α-1,2-fucosyltransferase that catalyzes the transfer of 1,2-linked fucose to galactose residue of glycans.
In this project, we aim to generate high throughput data that shows the regulation of FUT1 and FUT 2 by miRNA using miRFluR high-throughput assay. This assay utilized genetically encoded dual-color fluorescence reporters to identify regulatory miRNAs thus generating a comprehensive map of the regulation of FUT1 and FUT2 by approximately 2700 human miRNAs.
pMIR-3’ UTR sensors of FUT1and FUT2 have been developed. From our high throughput assay, we have identified 76 regulatory miRNAs for FUT1. For these regulatory miRNAs, the data have been validated using western blot, lectin binding assay, and q-RT-PCR experiments in multiple cell lines. To summarize, this study contributes to the understanding of the miRNA regulations of FUT1 and FUT 2 and their significance for normal biological function.

Sunday
3747539 - Kinetic mechanisms of isocitrate dehydrogenase 1 (IDH1) mutants | Poster Board #2717
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the NADP+ -dependent conversion of isocitrate to α-ketoglutarate (αKG). Mutations in IDH1, which primarily affect the R132 residue, can cause a neomorphic reaction where αKG is converted to D-2-hydroxyglutarate (D2HG). The catalytic efficiencies of D2HG production can vary greatly between IDH1 mutants. The arginine to histidine mutation at site 132, R132H, is one of the most common mutations identified in patients with lower grade gliomas and secondary glioblastomas and weakly drives D2HG formation. We have shown previously that smaller residues at residue R132, like R132G, drive more efficient D2HG formation. Here, we propose to study the kinetic features of an arginine to alanine mutation, R132A, an experimental mutation that is yet to be explored. Through steady-state kinetic analysis of the R132A mutation, we can gain a better understanding of what chemical and structural features can facilitate the neomorphic reaction.
Sunday
3747663 - Elucidating the kinetic mechanism of human METTL16 | Poster Board #2529
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Over 140 RNA modifications have been discovered, yet only recently have they been studied in-depth due to recent technological advancements. N6-methyladenosine (m6A) is an abundant RNA modification in messenger RNA (mRNA) and long non-coding RNA (lncRNA) that affects various cellular functions such as mRNA stability, phase separation of RNAs, and others. Methyltransferase-like protein 16 (METTL16) is one of four catalytically active m6A RNA methyltransferases in humans. Two well-known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3' untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3' triple helix of MALAT1. Using in vitro methyltransferase assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Our in vitro methyltransferase assays consist of purified recombinant human METTL16 (1-562) in combination with the U6 snRNA substrate and S-adenosylmethionine (SAM), the methyl donor, to initiate the reaction. Thus far, we have determined that METTL16 acts as a monomer when bound to either U6 snRNA or to the MALAT1 triple helix; METTL16 has an equilibrium dissociation constant of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent equilibrium dissociation constant for SAM with the METTL16-U6 snRNA binary complex is 112 µM. Under various assay conditions, the cancer-associated MALAT1 triple helix is not methylated by METTL16. Steady-state assays provided a kcat of 0.07 per minute and single-turnover assays established a kchem of 0.56 per minute. This difference in the rates suggests conformational rearrangements and/or product release may be rate-limiting. Mutations of the METTL16 K-loop structure led to a 7-fold increase in SAM binding to the METTL16-U6 snRNA binary complex. Future studies will focus on more METTL16 mutants, including critical residues in other regions and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16.
Sunday
3747955 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3748334 - Bile instigates structural destabilization and precipitation of MARTX, resulting in reversible activity inhibition of ACD and CPD effector domains | Poster Board #2533
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
While the primary function of bile in the gut is to solubilize fat, it also regulates the colonization of the gut microbiome. To counteract this, some bacteria have evolved to respond to bile by modulating the production of virulence factors. We speculate that due to their typically high conformational plasticity and low stability, virulence factors are easy targets for the denaturing activities of bile salts. If confirmed, toxin denaturation by bile salts may influence the pathogenic potential of bacteria and justify bile-related adjustments in the expression of virulence factors. In this work, we explored the effects of reconstituted bovine bile, sodium deoxycholate (DOCh), and sodium taurocholate (TCh) on the stability and activity of effector domains of multifunctional repeats-in-toxin (MARTX) from V. cholerae and A. hydrophila.
Protein integrity can be assessed by the accessibility of a protein’s intrinsic Trp fluorescence to collisional quenching. We noticed that intrinsic Trp fluorescence of different toxins is predominantly quenched by bile by increased by DOCh. Despite this, most toxins responded to both treatments by enhanced accessibility of Trp fluorescence to quenching by acrylamide. Next, the effects of bile and DOCh on toxin solubility were evaluated by pelleting. We observed increased precipitation of the toxins in the presence of DOCh. Effects of bile were milder and more discriminative, slightly promoting precipitation of some toxins, showing no effect on others, and even improving the solubility of yet others, e.g., actin crosslinking domain (ACD) toxin from psychrophilic A. hydrophila, when added above its melting temperature.
Upon delivery to the cytoplasm of host cells, MARTX domains are separated by activation of the cysteine protease domain (CPD). Cleavage of the CPD-containing four- and two-domain constructs was not affected by either DOCh or TCh but inhibited by whole bile. This inhibition was accompanied by the formation of high molecular weight protein species and could be reversed by the addition of a reducing agent, as revealed by SDS-PAGE under non-reducing conditions. Overall, these observations suggest that bile can compromise the structural integrity of the tested bacterial toxins, instigate their precipitation, and induce oligomerization via reversible cysteine oxidation, justifying the need for bacteria to adjust toxin expression levels.

Sunday
3748770 - Thermodynamics determines the coupling between growth and byproduct production | Poster Board #2509
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Engineered strains have been developed by knocking out specific genes to overproduce a valuable byproduct. The main concept is to remove reactions such that biomass production entails producing the byproduct. This way, we can use the growth rate as a selective pressure, where the mutants with higher growth have higher production capacity. Computational methods are proposed to find knockout strategies to couple growth and byproduct formation. However, none of these methods can integrate information about metabolite concentrations that play a key role in determining the directionality of the reactions. One of such methods is OptKnock, which is formulated as a bilevel optimization.
Here, we integrated thermodynamic constraints into the bilevel formulation of OptKnock to create TOptKnock. We showed that the computational efficiency of TOptKnock is on par with OptKnock. Due to the inclusion of thermodynamic constraints, TOptKnock can account for the metabolite concentrations. We demonstrated that the coupling between growth and byproduct formation might change in response to the variation in concentrations. As a result, a knockout strategy might be optimal for an intracellular condition but suboptimal for another. If metabolomics data is available, TOptKnock can search for optimal knockout interventions subject to the existing condition. We also envision that TOptKnock framework helps devise strategies for manipulating metabolite concentrations to couple growth and byproduct formation.

Sunday
3749274 - Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes | Poster Board #2916
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Chemo- and regio-selective catalysis of C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-oxo-glutarate (2OG) dependent non-heme iron halogenases catalyze selective chlorination/bromination of C-H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well-understood. In this talk, we show that tyrosine placement in the SCS of non-heme iron halogenases have a huge impact on their structure, function, and reactivity. We discover that a tyrosine mutant (F121Y) in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies show that the DOPA modification in SyrB2 renders the enzyme non-functional. Further bioinformatics analysis suggests that halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Overall, this presentation will demonstrate the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases.
Sunday
3749321 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3749412 - Photo-controlled serotonin receptor modulation as a model for neuron signaling using non-neuronal cell lines. | Poster Board #2517
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Spencer Kim, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Despite an abundance of interest, studying factors that influence neuron signaling remains a challenging field of research. There are several factors that limit researchers from studying neurons, including rapid signaling kinetics, cell health sensitivity, and lack of primary neuron availability. To address these issues, we have developed a model for neuron signaling that uses immortalized non-neuronal cell lines as the biological substrate and non-invasive photocontrol as the signal stimulant. Here, we demonstrate the systems use as an effective model to phototemporally regulate intracellular calcium on a timescale relevant to neuronal calcium signaling.
Sunday
3749953 - Determining the biochemical and cellular basis of STING activation | Poster Board #2804
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Because DNA is the genetic blueprint of life, it is hard to imagine that it can also act as a damage- or pathogen-associated molecular pattern (DAMP/PAMP) to trigger immune responses. Different cellular stresses or infections lead to the release of DNA into the cytoplasm, which is considered a danger signal indicative of pathogen invasion. One of the most important sensors of cytosolic DNA is the cGAS-STING pathway, which triggers protective responses that are important in contexts like infection or cancer, although excessive activation of this pathway can be harmful and contribute to inflammatory responses. While unique signaling events are important for STING activation, it is still unclear exactly how these essential events mechanistically lead to STING activity. STING polymerization, trafficking and palmitoylation have all been shown to be important for STING signaling, but whether one event depends on another and exactly how each event promotes the signaling cascade remains to be understood. Therefore, developing an in-depth understanding of the regulation mechanisms of the STING pathway will elucidate how it can be positively or negatively regulated and consequently, how it can be dysregulated in different disease contexts. Here, we harness our molecular toolkit to glean biochemical and structural insight into the importance of each STING signaling event, and hopefully shed light on the most promising avenues by which STING should be pharmacologically inhibited in order to better inform the design and efficacy of next generation STING modulators for treatment of various diseases.
Sunday
3749957 - Chemical optimization and mechanistic investigation of small-molecule agents for long-term ex vivo expansion of granulocyte-monocytes progenitors | Poster Board #2612
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Granulocytes and macrophages, the two major components of the innate immune system, are immune cells with tremendous therapeutic potentials to combat infections and cancer. For example, macrophages have the ability to target and eliminate cancer cells via phagocytosis while granulocytes such as neutrophils can effectively treat neutropenia and decrease the risk of life-threatening infections, notably in cancer patients undergoing chemotherapy. These cellular therapies require large amounts of granulocyte and macrophage; however, there are no good methods for expanding granulocytes and macrophages that are terminally differentiated while the major source of these cells, namely the bone marrow and cord blood, fails to provide sufficient amounts of cells for therapeutic applications. The granulocyte-monocyte progenitor (GMP) is the common progenitor of granulocytes and macrophages. We previously discovered that a culture condition consisting of a cytokine, a kinase inhibitor, and a small molecule named SKL2001, enabled long-term expansion of mouse GMP, but not human GMP, ex vivo. Herein, we report the chemical optimization of SKL2001 to allow for efficient expansion of human GMP and our efforts to identify the molecular target of SKL2001 responsible for GMP expansion. Through a structure-activity relationship study of SKL2001 containing more than 100 analogues, we discovered several analogues that significantly improved human GMP expansion compared to SKL2001 at even lower concentrations. In particular, compound TN-2-30 could expand human GMPs for up 60 days while retaining the ability to efficiently differentiate into phenotypic and functional granulocytes and macrophages. Additionally, we derivatized SKL2001 with chemical crosslinking groups to allow for the identification of molecular target(s) of SKL2001 responsible for GMP ex vivo expansion. The ability to perform long-term expansion of the progenitors would open the door to investigate and unleash the therapeutic potentials of granulocytes and macrophages. Moreover, identification of molecular targets for GMP expansion would provide monumental insights into the self-renewal ability in other types of stem or progenitor cells, allowing their implementation for different purposes.
Sunday
3750033 - Indirect quantification of 5-hydroxymethylcytosine (5-hmC) | Poster Board #2607
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Epigenetic modifications of DNA have been a hot topic for a few years now, especially about the methylation of DNA. While the 5-methylcytosine (5-mC) modification of DNA is being thoroughly explored by Horvath and other people, the rest of the oxidative chain (5-hydroxymethylcytosine (5-hmC), 5-formylcystosine and 5-carboxylcytosine) has not been explored as much. These products are obtained with the oxidation of 5-mC by the ten eleven translocation (TET) enzyme. Given the already important discoveries of 5-mC with epigenetic clocks and its roles in many diseases, it is urgently needed to study the other modifications, and for that purpose find methods to quantify these. Hereby we present a study about a simple indirect quantification of 5-hmC, where 5-hmC is first oxidized to 5-foC before attaching a linker based on a biotin tag linked to an aminooxy motif, thus forming a very stable oxime bond. The biotin tag can then be used to quantify the amount of 5-hmC in the cell with its very strong binding to streptavidin. As a cost-efficient chemical method that does not rely on PCR enrichment or enzymes, it allows for choices in detection incorporating a biotin tag. The overall significance makes an impact on the scientific community as increased knowledge about the role of 5-hmC and its implications in diseases has become paramount.
Sunday
3750114 - Soft hydrogel microbial modulators and biological applications | Poster Board #2601
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Soft hydrogel-based architectures which allow the diffusion of solutes across the hydrogel matrix have been utilized to develop various functional systems by confining microorganisms. Recently, new hierarchical hydrogel structures have been pioneered to create multi-layered soft hydrogels in a concentric fashion to provide fundamental insights at the molecular level. Yet the strategies might not be standardized because different design considerations are required due to a wide range of chemical processes involved. At small scales, the fabrication of micro-size architectures requires higher reaction rates than that of macroscopic objects, which causes agglomeration between gels in a reaction bath. Only a few methods have been introduced to produce a single multi-layered capsule with diameters between 4 and 20 mm. Even with predictive mechanisms in various hydrogels, challenges exist in both mass production and scaling-down of the size of hydrogel architectures, and multi-layered techniques for cell encapsulation studies are even sparse.

To address the challenges, we have developed a biocompatible model with robust design, utilizing alginate-based core-shell architectures. Herein, technical obstacles to create confined architectures for effective bacteria confinement have been resolved to retain functionally engineered cells inside the hydrogel architectures without disruption of the structures. Numerous small ecological systems in a hydrogel-based environment can be generated by recapitulating specific bacteria inside the conceived architecture. Microbial colonization occurs, ensuring cell longevity and production of molecules to serve as independent microbial modulators with direct or indirect activation of a response regulator by stimuli, including signaling molecules. To show the applicability of the platform, further, we demonstrate a reliable strategy with hydrogel-based confinement techniques to evaluate the concept of artificial natural habitats in developing bacteria-based functional systems (i.e., dynamic communication models, bacteria-mammalian cell interaction system, enzymatic bioreactors, etc.). We believe this approach has significant potential not only to study intra/multicellular complex communications in a highly specific manner but also to design a soft hydrogel-bead functional system.

Sunday
3750241 - Penta-fluorination of Phe-Phe motif to overcome homochirality in hierarchical self-assembly of hydrogels | Poster Board #2519
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Biomedical research is at the forefront of medicine, and the hydrogel materials which are formed from nanostructures are a key part of this research as it progresses through the twenty-first century. The hydrogel material provides biomedical researchers with the best tool for the simulation of in vivo extracellular conditions for the in vitro study of cells. This study focuses on the creation of dipeptide-based hydrogels by modifying the well studied diphenylalanine motif. One issue that arises in the production of dipeptide-based hydrogels is the stereochemistry of the R groups on the dipeptides that form the nanostructures. Previous research on the diphenylalanine motif has shown that the nanostructures that are formed from homochiral dipeptides resulted in a material that is unstable and over time becomes unusable. This instability occurs from the stereochemistry of the homochiral diphenylalanine as it forces the aromatic rings into a perpendicular orientation within the nanostructure, instead of the regular parallel orientation that allows for π-stacking with heterochiral diphenylalanine. To further assess this phenomenon, we used fluorinated phenylalanine to determine if it could overcome the molecular orientation issue of the amino acids by increasing the strength of intermolecular forces and inducing a rotation of the sigma bond. The induced rotation of the sigma bonds resulting in a new orientation of the molecules will allow for stable nanostructures and eliminate the need to account for stereochemistry in the production of dipeptide-based hydrogels.
Sunday
3750318 - Paper-based purification and concentration of genomic DNA using isotachophoresis | Poster Board #2914
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

In recent years, much work and effort have been put into developing affordable and simple POC-nucleic acid amplification tests (NAATs) for diagnosing infectious diseases like tuberculosis. While the primary focus has been on nucleic acid (NA) amplification and detection strategies, many POC-NAAT assays still rely on complex sample preparation methods. To overcome this limitation, we demonstrate a POC-compatible paper-based device capable of purifying and concentrating Mycobacterium tuberculosis genomic DNA (gDNA) from viscous mock-sputum using isotachophoresis (ITP) within 15 min. ITP is an electrokinetic separation and concentration technique consisting of a fast-moving leading electrolyte (LE) and a slow-moving trailing electrolyte (TE). It is used to concentrate the charged species (DNA) at the LE/TE interface. The paper-based ITP device consists of an 11-layered foldable structure with circular disks of Whatman filter paper sandwiched between the two reservoirs (containing TE and LE buffer) that forms a paper channel for the liquid to flow. gDNA (102-100 copies/µL) mixed with 900 µL of TE buffer (TTD) was added to the TE reservoir along with simultaneous addition of 900 µL of LE buffer (TH) to the LE reservoir. A voltage bias of 18 V was applied between the platinum electrodes in the reservoirs. Post-ITP, DNA gets concentrated in some of the initial paper disks. Each paper disk can be used as a PCR/real-time PCR template.
The device was able to concentrate and purify the DNA even in the presence of inhibitory molecules present in the mock-sputum. qPCR results showed approx. 13x concentration of purified DNA, post-ITP. This low-voltage paper-based ITP platform is the first demonstration of sputum DNA preparation using paper-based ITP. It offers a critical pre-analytical step of gDNA from complex sputum samples and complements paper-based isothermal nucleic acid amplification TB tests. In another set of experiments, we also demonstrate the device's capability to electrochemically lysis M. Smegmatis bacteria, spiked in mock sputum, and simultaneous purification and concentration of the Msm gDNA into the paper disks. Ultimately, we aim that these tools would enable molecular diagnostics of TB or any other infectious disease in low-resource settings.

Sunday
3750760 - Using a hybrid input-output algorithm for ab initio phase retrieval of high solvent content protein crystals | Poster Board #3016
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

It is well understood that the crystallographic phase problem is an obtrusive bottleneck in the workflow for protein structure determination using direct methods. Whereas traditional phasing protocols require prior known structural information, here we present results from a hybrid input-output (HIO) algorithm that can phase high solvent content proteins directly from X-ray diffraction data–that is, ab initio. This HIO phasing method can successfully reproduce the electron density maps of several previously resolved structures in the protein database (PDB) using only the experimental reflections recorded during diffraction. The test set used in this study included a combined total of 45 proteins in space groups P43212 and P212121. Successful algorithm convergence was observed for 16 of these proteins. Thereafter, we probed the sensitivity of certain HIO input parameters and considered how these may be optimized to yield an improved frequency of convergence. This direct method of phase retrieval is a desirable alternative in cases where experimental phasing is not biologically feasible or in cases where too few structural analogues have been resolved.

Sunday
3750844 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3751593 - ENPP1 is an innate immune checkpoint by degrading extracellular cGAMP | Poster Board #2527
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
ENPP1 is the dominant hydrolase of extracellular cGAMP in the STING pathway but has rich cGAMP-STING-independent pathophysiology. Mounting evidence shows that ENPP1 has pro-cancer roles with unclear mechanisms. Using ENPP1 genetic knockout in a murine breast cancer model, we showed that ENPP1 dampens immune infiltration and activation. In parallel, specific depletion of extracellular cGAMP from the tumor microenvironment yields a similar immune signature. Furthermore, using a genetic knock-in mouse harboring an ENPP1 mutation specifically defective towards cGAMP hydrolysis, we demonstrated that ENPP1’s cGAMP degradation activity alone is sufficient to facilitate tumor progression. ENPP1 on both cancer and normal tissues contributes to its innate immune checkpoint activity and should be blocked to turn cold tumors hot. Finally, ENPP1 activity levels vary in mouse tissues and human donors and the common K173Q allele harbors markedly increased activity, a discovery with profound implications in patient selection for ENPP1-based cancer immunotherapy.
Sunday
3751902 - *In vitro* Screening of adenylate cyclase inhibitors in cyclic di-GMP pathways | Poster Board #2720
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Pseudomonas aeruginosa is a biofilm-forming, gram-negative bacteria widely known for its high antibiotic resistance in chronic infection models, such as cystic fibrosis lung infections and ventilator-associated pneumonia. Biofilms initiate when bacteria attach to a surface with extracellular appendages, forming a monolayer, progressing to form sessile macro-communities. This motile to sessile transition is facilitated by the secretion of the extracellular polymeric substances (EPS), encapsulating the bacteria. This EPS network forms a protective barrier over bacteria, making it insusceptible to host defense mechanisms used to treat respiratory infections. Cyclic di-GMP is a second messenger, signaling nucleotide molecule that directly modulates biofilm formation, virulence, and motility in bacteria. It is synthesized from two GTP molecules by diguanylate cyclases (DGCs) and is hydrolyzed by phosphodiesterases (PDEs) to form linear pGpG. Diguanylate cyclases and phosphodiesterases comprise a GGDEF domain and an EAL/HYF domain, respectively, responsible for the degradation of cyclic di-GMP. Bacterial regulation of DGCs and PDEs is critical for biofilm formation and maintenance. Our goal is to repurpose a set of cyclic-AMP derivatives, already shown to inhibit cyclic AMP cyclases, as bifunctional cyclic nucleotide inhibitors to offset complete bacterial cyclic nucleotide regulation by testing their binding affinities and bioactivities against these enzymes. This theory suggests that cyclic-AMP analogs can eradicate biofilm formation in response to changes in adenylate and diguanylate cyclase activity, catalyzing the transition from an antibiotic resistant, sessile state to an antibiotic susceptible, planktonic state in P. aeruginosa, as well as other biofilm forming bacterial pathogens.
Sunday
3752117 - The role of W106A in the communication between the promiscuous allosteric site and the active site in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase | Poster Board #2721
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The synthesis of glycogen in bacteria is regulated by ADP-glucose pyrophosphorylase (ADP-Glc PPase), which catalyzes the reaction between glucose 1-phosphate (Glc1P) and adenosine triphosphate (ATP) to form ADP-glucose (ADP-Glc) and pyrophosphate (PPi). In Agrobacterium tumefaciens, ADP-Glc PPase is activated by fructose 6-phosphate (Fru6P) and pyruvate and inhibited by AMP. In Escherichia coli, it is activated by fructose 1,6-bisphosphate (FBP) and inhibited by AMP. Based on previous studies a loop consisting of a span of residues in the ADP-Glc PPase from E. coli was identified as important to the allosteric activation pathway of the enzyme. Trp113 is part of this loop. When mutated to alanine, it showed no response to the allosteric activator FBP. It was found that Trp113 is highly conserved since it was present in all known ADP Glc-PPases. Trp106 was identified as the homologous residue in A. tumefaciens. When mutated to alanine it had no response for the activator Fru6P but in the presence of pyruvate, the enzyme behaved like the wild type (WT). Given that the ADP-Glc PPase from A. tumefaciens has two major different activators, E. coli has one and they both share the same inhibitor, the promiscuity of the allosteric site was investigated. The sugar phosphates studied had similar structures to the native activators of the enzyme. We found that these effectors fit into two categories based on their behavior, some as activators and others as inhibitors. Given the results of both studies, we analyzed how each of these molecules behave in the mutant W106A. The effectors exhibited similar results to the WT enzyme in the form of two distinct groups, activators, and inhibitors. None of them were able to activate the mutant, but they were able to bind indicating that the allosteric signal was disrupted. Here all the molecules exhibited an increase of activity at around 1.2- compared to the 9-fold displayed in the WT. The molecules that inhibited had a remaining activity of around 1.3% and in the WT, it ranged from 4-12% meaning that there was a larger difference between the initial activity (V0) and the final activity (V). The results from these studies demonstrate that the communication within the enzyme is different for both regulators since pyruvate was still able to activate W106A. It also reinforced the fact that Fru6P has a different allosteric site than pyruvate and that these molecules are all binding to the same site.
Sunday
3752356 - Functional characterization of the NosP associated histidine kinase, NahK, and its role in biofilm regulation in Pseudomonas aeruginosa | Poster Board #2531
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Pseudomonas aeruginosa is a biofilm-forming, gram-negative bacteria that is the main causative agent of lung infections in patients with cystic fibrosis. Biofilms are multicellular communities formed when bacteria attach to a surface and secrete a thick, mucoid protective barrier known as the exopolymeric substance, which is mostly composed of polysaccharides extracellular DNA, and proteins. Due to these biofilm properties, P. aeruginosa has been classified as a number one priority in need of a new antibiotic/treatment by the World Health Organization. It has previously been shown that the diatomic gas, nitric oxide (NO), mediates biofilm dispersal. Our lab investigates the NO sensing protein (NosP) and its co-cistronic associated histidine kinase (NahK). NosP has previously been shown to induce biofilm dispersal by inhibiting the kinase activity of NahK. In P. aeruginosa, NahK has been shown to phosphorylate a histidine phosphotransfer protein (HptB), which acts a phosphorelay hub for multiple, complex two component signal transduction systems. However, preliminary data has revealed several observed phenotypes independent of direct HptB signaling; thus, suggesting the involvement of NahK in other signaling cascades. In P. aeruginosa, NahK is a cytosolic, sensory hybrid histidine kinase containing three uncharacterized N-terminal PAS signaling domains. We hypothesize that the signaling domains provide a secondary regulatory system over kinase activity, separate from the NO-mediated regulation through NosP, resulting in biofilm dispersal through the downstream effects of the HptB phosphorelay system and secondary signaling cascades.
Sunday
3752411 - Expression and purification of the ligand of ATE1 (Liat1) from Mus musculus | Poster Board #2537
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The proteome is large and dynamic chiefly due to post-translational modifications (PTMs);however, some PTMs such as arginylation are understudied. Arginylation is the post-translational addition of the amino acid Arg to a eukaryotic protein, and this process is catalyzed by the highly-conserved enzyme arginine transferase (ATE1). Post-translational arginylation is essential for normal eukaryotic development, but much remains unknown regarding the mechanism and the regulation of this process. A recently discovered interaction partner of ATE1 is the ligand of ATE1 (Liat1), but the structure and the function of this protein are both enigmatic. In this work, we cloned, overproduced, and attempted to purify mouse wild-type and truncated Liat1 proteins for structural and biophysical studies. While Liat1 can be heterologously overproduced in large quantities, solubility issues required re-folding attempts. On-column and dialysis-based refolding techniques are described. Additionally, we used protein structural modeling to predict the structure of the region of Liat1 that is not intrinsically disordered. These promising results provide the insight into the structure and the function of the first-known protein partner of ATE1.
Sunday
3752531 - High throughput screening of immunomodulators for vaccine adjuvants | Poster Board #2535
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Adjuvants, molecules that boost immunogenicity, are often necessary for innate immune system stimulation in both vaccinations and immunotherapies. Adjuvants typically target pattern recognition receptors (PRRs), resulting in the activity of two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway and the interferon regulatory factors pathway (IRF). Engineering the immune response via fine control of these pathways, however, is quite difficult. We have identified an array of small molecules that modulate the NF-κB/IRF activity of several PRR agonists through a multi-step high throughput screening approach. Instead of developing new agonists, we demonstrate the ability to alter existing responses with immunomodulators, tailoring the activity to a desired need. Modulation enhances or inhibits innate pathways. The effect can be general altering responses to multiple PRRs, or specific changing as few as just one receptor response. Obtaining a new method of that offers greater control over early immune activation provides researchers with a new tool for various applications from prophylactic vaccines to therapeutic treatments.
Sunday
3752658 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Session Type: Poster - In-person

Sunday
3752697 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3752721 - Unlocking new reactivity of UstDv2.0 using protein engineering for the synthesis | Poster Board #2600
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon–carbon bond-forming transformations in traditional organic chemistry and nature, relatively few C–C bond-forming enzymes have found their way into the biocatalysis toolbox. Previously, we successfully engineered the enzyme UstD to produce an activated catalyst, UstDv2.0, capable of performing a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard γ-hydroxy amino acids. New engineering efforts on UstDv2.0 have continued to expand the electrophile scope from aldehydes to include various ketones.
Sunday
3752745 - Development and characterization of fluorescent chemical tools to study human carboxylesterase 2 (CES2) | Poster Board #2602
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The two major human carboxylesterases (CESs), CES1 and CES2 play a key role in the hydrolysis of xenobiotic esters including aspirin, methylphenidate (Ritalin), and the illicit narcotics cocaine and heroin. CES-mediated metabolism of ester containing drugs can result in their activation or deactivation and enhance clearance. Despite the established role of CESs in drug metabolism, few techniques exist to study their activity in live cells. Fluorescent chemical tools have been developed to be able to expand the methods used to study CES activity, however, many of these tools are not sufficiently characterized to be able to confidently study CES activity in live cells. This is particularly true for CES2 where substrate specificity can overlap with other cellular esterases. To address this issue, we have worked towards identifying and developing new fluorescent chemical tools to study CES by evaluating candidate chemical tools for their specificity for CES2 over CES1 in vitro and in live cells using small molecule inhibitors and shRNA. Overall, our studies will result in full characterization of fluorescent chemical tools for studying human CESs and will lead to improved methods for studying the role of CESs in human drug metabolism.
Sunday
3752762 - Potential antibacterial properties of green synthesized copper nanoparticles | Poster Board #2604
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Developing ecologically friendly methods for metal nanoparticle synthesis is at the forefront of current research efforts, including use of plant extracts as green reducing agents. Copper nanoparticles will be synthesized using Spinacia oleracea (spinach) leaf extract as a reducing agent, as related plant species have been shown to reduce copper(II) ions to copper nanoparticles. Metal nanoparticles have shown promise as potential antibacterial agents in medical implant coatings, wound care materials, and antibiotic delivery tools. Therefore, the potential antibacterial properties of the synthesized copper nanoparticles will be investigated against Escherichia coli using disc diffusion method. It is expected that if the synthesized copper nanoparticles exhibit antibacterial activity the zone of inhibition will show a concentration dependence; namely, the zone of inhibition will increase with an increase copper nanoparticle concentration.
Sunday
3752811 - Uncovering the mechanism of extracellular cGAMP signaling in T cells | Poster Board #2608
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Double-stranded DNA (dsDNA) can appear in the cytosol during infection or cellular damage. The cell can recognize cytosolic dsDNA through the enzyme cyclic-GMP-AMP synthase (cGAS), which catalyzes the formation of the cyclic-GMP-AMP (cGAMP) that triggers signaling cascades to fight off infection or clear aberrant cells. Previously, we discovered that many cancer cells upregulate cGAMP production and secrete it out of the cell. Extracellular cGAMP is then selectively taken up by surrounding cells to activate them. For some immune cells, extracellular cGAMP uptake leads to better tumor clearance. For T cells, however, cGAMP signaling leads to cell death, which is detrimental to their anti-cancer effects. Therefore, it is important to understand how extracellular cGAMP signaling is regulated in T cells. We previously showed that cells use a mix of protein transporters that facilitate cGAMP import or export across the cell membrane. We have identified several cGAMP transporters used by human peripheral blood mononuclear cells and derived macrophages, and vasculature cells. However, the T cells transporter that permits cGAMP mediated T cell killing has not been identified. Using a genetic knock-out screen, here we report a potential cGAMP transporter in T cells. This novel transporter can hopefully be a target for therapeutic inhibition to improve T cell response in cancer therapy.
Sunday
3752893 - Mechanistic evaluation of tautomerases-catalyzed formation of malonate semialdehyde from acetylenecarboxylic acid | Poster Board #2606
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Decarbonization of the economy will require increased utilization of one-carbon chemicals such as carbon dioxide (CO2) and methane (CH4). Acetylenecarboxylic acid (ACA) can be synthesized from methane and carbon dioxide. Our goal is to transform ACA into value-added chemicals such as malonic acid and 3-hydroxypropionic acid. Here we investigate two tautomerase enzymes, cis-CaaD and Cg10062, in the utilization of ACA for the production of malonate semialdehyde (MSA), a common intermediate of malonic acid and 3-hydroxypropionic acid synthesis. These enzymes have 53% similarity, identical active site amino acid residues, a homologous β-α-β structure, a catalytic proline at the N-terminal, and require no cofactor or metal for catalysis. cis-CaaD catalyzes the formation of MSA as the nearly exclusive product (Km 1160 μM), while Cg10062 affords acetaldehyde (ACH, 81% product formation) as the major product with low amounts of MSA (19% product formation, Km 66 μM). In our ongoing efforts to improve the rate of formation while preserving exclusive MSA production, we have carried out site-specific mutagenesis and protein crystallography to understand the subtle differences between these two enzymes. Here, we present the kinetic characterization of multiple variants of both cis-CaaD and Cg10062 as well as their substrate-soaked atomic resolution crystal structures that may give insight into both the mechanism of reaction and the relative products formed for each variant.
Sunday
3753071 - Electrostatic interaction promotes aggregation of novel tau fragment | Poster Board #2920
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Tau, a microtubule-associated protein, promotes tubulin self-assembly into microtubule polymers, which helps to maintain the integrity of the axonal transport tract. Tauopathies are disorders marked by aberrant intracellular tau aggregation in the brain. In this study, we employed a 20-residue short peptide sequence (298–317) generated from the longest tau isomer (2N4R). The kinetic investigation of the fragment in the presence of thioflavin T (ThT) showed that the peptide was extremely soluble in a physiological buffer, even at very high concentrations (up to 400 µM). CD data revealed that the tau fragment contained over 50% random coil structures, with minima at ∼200 nm. Then we studied the fibrillation process of the fragment in the presence of polyanion Heparin. The addition of Heparin promoted the aggregation of the tau fragment. The fluorescence intensity of the ThT signal rose linearly with the increase in tau concentration, which emulated the previous result with the tau (containing four repeats, K18). We calculated lag time, t50, and Kapp through curve fitting in the Boltzmen distribution. Results showed that the higher the concentration of tau fragment, the shorter the lag time, t50, and higher Kapp. In CD studies, this novel tau fragment, in the presence of Heparin, converted into stable secondary structures with β-sheet as its major conformation. Later, the presence of fibrils was confirmed by the AFM studies. Finally, we assessed how tau fragment interacted with anionic Heparin. Interestingly, aggregation of the novel fragment at 12.5 µM (with 1.5 uM Heparin) was highly sensitive to salt. The presence of salt (NaCl, 300 mM in 50 mM PB) completely suppressed the ThT signal. Ionic salt mediated inhibition of the aggregation suggesting that the peptide interaction with the heparin was electrostatic in nature. Therefore, suppressing the electrostatic interaction between tau and Heparin could lead to the development of tau fibrillation inhibitors.

Sunday
3753142 - Assessing a monovalent degrader of CK1α in ovarian cancer models | Poster Board #2610
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Ovarian cancer is the most lethal gynecological malignancy in the United States, with less than half of newly diagnosed women surviving for more than five years. In the absence of effective screening measures, ovarian cancer is often diagnosed in an advanced stage rendering the disease difficult to treat successfully. Despite efforts to develop targeted therapies, the standard of care remains debulking and platinum-taxane based chemotherapy, demonstrating the need for both new therapeutic targets and targeted therapies. Casein kinase 1 alpha (CK1α) plays a key role in several cellular pathways implicated in cancer, such as the Wnt/β-catenin, NF-κB, and p53 apoptotic pathways. CK1α is an established and important therapeutic target in hematopoietic malignancies and may be a potential therapeutic target in the context of additional malignancies, such as ovarian cancer. Recently, we developed a monovalent degrader of CK1α that exhibits low nanomolar potency. Here we describe the assessment of this degrader in the context of tissue culture cell lines derived from several solid tumors, including ovarian cancer, and we demonstrate the potential application of CK1α degradation as a therapeutic modality in the treatment of ovarian cancer.
Sunday
3753419 - Predicting binding affinities of nucleic acid ligands with free energy perturbation | Poster Board #2616
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Accurate prediction of ligand binding affinities has been of crucial importance for medicinal chemists. Among various methods for calculating binding affinities, free energy perturbation (FEP) calculations have emerged as one of the most reliable and rigorous approaches. However, the application of this method has overwhelmingly been focused on drugs binding to proteins. Yet with their important roles in essential cellular processes, such as cellular reproduction and protein biosynthesis, nucleic acids have emerged to play an equally important role in drug discovery. Specifically, DNA binders alter gene expression and RNA binders are able to disturb the transcription and translation processes. As such, nucleic acids are potential targets in anticancer, antiviral, and antibacterial drug discovery programs. Here, we apply our FEP technology (FEP+) to estimate the binding affinities of small molecules to nucleic acids. We provide a systematic characterization of FEP+ in predicting relative binding free energies of congeneric ligands binding to a variety of DNA and RNA structures, such as single and double stranded helices, g-quadruplexes, loop motifs, as well as nucleic acid-protein complexes. We will highlight the successes and detail potential challenges in using FEP+ to predict binding free energies of small molecules to nucleic acids, while expanding the domain of applicability of FEP+.
Sunday
3753427 - Fingerprinting small molecule modulators of nucleolar ribosome biogenesis | Poster Board #2618
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Nucleolus is a membraneless and multicompartmentalized nuclear body governing ribosomal RNA (rRNA) biogenesis, a fundamental process that enables protein synthesis in all living organisms and is misregulated in various diseases. The characterization of several known chemotherapeutic drugs as modulators of distinct rRNA biogenesis stages and associated nucleolar morphologies has challenged the accepted mechanisms of action for these compounds and simultaneously established the nucleolus as an attractive therapeutic target. Nevertheless, there are currently no comprehensive and quantitative frameworks that capture and relate drug-induced changes in nucleolar morphology and its liquid-like material properties to its function. We hypothesized that we can establish such a structure-function framework by screening a panel of well-characterized nucleolus-perturbing small molecules and measuring changes in biophysical properties of the nucleolus in cells from microscopy images. Here, we combine morphometric, thermodynamic, and kinetic rRNA flux measurements to reveal “fingerprints” for drugs that modulate distinct stages of rRNA biogenesis. Preliminary insights reveal how perturbing specific non-equilibrium nucleolar processes maps to differential thermodynamic signatures of select nucleolar proteins to in turn lead to unique morphological changes of this condensate. We anticipate that this framework provides a starting point for understanding and predicting structure-function relationships from nucleolar phenotypes as well as for developing high-content screening platforms to expedite the discovery of novel nucleolus-targeting therapeutics.
Sunday
3753516 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3753581 - Biomarker-free diagnostics using machine learning-guided optical nanosensor arrays | Poster Board #2901
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Christopher Smith, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Recently, the integration of machine learning and nanotechnology has led to several advances in many fields including agriculture and medicine. However, the intersection of machine learning and nanotechnology has yet to breach its surface of potential. Machine learning has proven to be a useful tool for biomedical diagnostics. However, there is no artificial intelligence that can perform biomarker-free detection using experimental results. Here, we demonstrate a workflow using our algorithm termed AGONS and optical nanotechnology platform for biomarker-free detection of various biological systems including proteins, cancer cells, miRNA mimics, and more. Previously, we had assembled a nanosensor array that achieved 70-90% predictive accuracy against these targets using simple chemometric modeling. However, harnessing machine learning has allowed us to tremendously raise the accuracy to nearly 100% while reducing overall nanosensor array size. Utilizing AGONS provides two advantages: (1) automatic design and guiding of nanosensor array development through feature extraction techniques and (2) improved predictive accuracy. These combined advantages allow for improved downstream data collection for robust model development, sensor cost by the reduction in array size, and wider implementation space for array-based diagnostics. Through AGONS guidance in nanosensor array design, new opportunities for biomarker-free detection of the most challenging biological targets are possible.
Sunday
3753625 - Generating modified aptamer libraries through targeted mutations | Poster Board #2626
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Nucleic acid aptamers have emerged as flexible, tunable, and mass producible receptors for a wide range of targets and potential theranostic applications. A remaining bottleneck to wider use of aptamers is their low functional group diversity compared to antibodies. We report a strategy for making modified aptamer libraries through targeted mutations to critical residues of existing aptamer scaffolds. Each mutation is randomized to one of three standard nucleobases, adenine (A), cytosine (C), or guanine (G) or a synthetic thymine analogue (X), which bears an abiotic group. The resulting library members have the advantages of being functionalizable through click chemistry, completely covering the desired sequence space, adopting structures that are widely used in existing aptasensor platforms, and displaying enrichment after a single round of competitive selection.
Sunday
3753679 - Structure and specificity of the GT-B glycosyltransferases from Campylobacter concisus | Poster Board #2630
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Glycoconjugates represent a class of macromolecules relevant for bacterial viability and virulence, as they play a key role in host adherence. Despite the capability to synthesize a diverse set of glycoconjugates, bacteria share common mechanisms of biosynthesis. The pathways involve transfer of a sugar molecule from a nucleotide activated sugar donor to a lipid acceptor polyprenol phosphate (Pren-P). The glycan-based macromolecules are diversified through the action of glycosyltransferases, which vary in their donor-molecule selectivity. Understanding the factors influencing selectivity of glycosyltransferases lays the basis for the development of novel selective antibiotics. A majority of GTs in the pgl operon of C. jejuni, C. concisus, C. fetus, and other Campylobacter members, namely PglA, PglJ, and PglH, belong to the GT-B superfamily in a subclass of membrane-associated (“monotopic”) proteins. The GT-B type fold is characterized by two β/α/β Rossmann-fold domains with the catalytic center located in a large cleft between them. In this work we report the first structural characterization of the glycosyltransferase PglA, from Campylobacter concisus ATCC 33237, as well as identification of its preferred substrate via nano differential scanning fluorimetry (nanoDSF) screening.
Using X-ray crystallography, we have determined a 1.9 Å structure of PglA, bound to its native donor substrate, uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc). Guided by this structural information we designed a series of site-directed variants to gain understanding of the factors influencing PglA substrate specificity. The substrate of the next enzyme in the pathway, PglJ, has been identified as a 217 Da sugar in the literature, based on its mass spectrometry fragmentation pattern, presumably N-acetylhexosaminuronic acid of unknown stereochemistry. Herein, we report nanoDSF and luminescence based activity assay results showing that the C. concisus ATCC 33237 PglJ, reacts preferentially with UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) over its epimer, UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA).

Sunday
3753840 - Stopping intracellular viral replication and immune system escape using CholestosomeTM technology | Poster Board #2632
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
High viral load is a key driver in the progression of the many viral diseases, from influenza to Covid-19 and HIV, contributing to an increased likelihood of latent infections and severe complications leading to death. Most therapeutics target extracellular virus. Intracellular virus escapes this treatment leading to treatment resistant long term and latent infections. Targeting the intracellular virus fraction could thus reduce or eliminate long-term consequences of viral disease. This laboratory has developed a patented platform-based drug delivery technology using only cholesteryl esters, called a Cholestosome. Cholestosomes can encapsulate a large variety of molecules and can confer a unique oral bio-availability to peptides and proteins as well as demonstrated intracellular delivery. The objective of this study was to use this delivery system to provide intracellular delivery of Zn2+ and antiviral antibodies to inhibit viral replication. Zn2+ is a known inhibitor of viral replication but intracellular movement of Zn2+ is tightly regulated. Cholestosome were used to encapsulate and deliver Zn2+ and anti-COVID-19 antibodies into A549 cells infected with the betacoronavirus OC43. Encapsulated Zn2+-treated OC43 infected cells showed a 60% reduction in virus caused cell death. Encapsulated antibodies show similar outcomes. An effective oral therapeutic delivery modality that does not require challenging storage conditions, Cholestosomes provide a rapidly deployable solution to the problem of proliferative viral infection. The ability of Cholestosome vesicles to provide rapid delivery of a precise dose of an antiviral agent directly into cells would close a major therapeutic gap in the current treatment.
Sunday
3753844 - Metals in cancer chemotherapy: The prospect of trans-platinum compounds | Poster Board #2634
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Moses Olusegun, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Platinum-based anticancer agents are among the most widely accepted metal-based chemotherapeutics with cisplatin, despite its profound drawbacks, such as severe toxicity, lack of selectivity, inherent and acquired resistance, and transient retention in the bloodstream, still the most clinically and universally used metal-based anticancer agent. Several research efforts have been targeted towards developing alternative platinum-based cytostatic agents that will not only eliminate some of the challenges that are associated with cisplatin but also improve on its cytotoxicity. Most of the synthesized cis-configured complexes, which comprised of mainly the second-generation platinum-based drugs, such as carboplatin, oxaliplatin etc., are yet to be globally accepted and moreover, many of them are still encumbered with some of the limitations that are plaguing the continued use of cisplatin. Among the complexes that are currently being considered by medicinal bioinorganic chemists, platinum(II) complexes with trans-stereochemistry are attract a lot of research interest. This is because the complexes were found to exhibit pharmacodynamics that are relatively complex and radically different from those already established for cisplatin and its derivatives. The initial thought amongst many medicinal bioinorganic researchers that cis-configuration is a prerequisite for cytotoxicity because of the dearth of activity of the non-toxic transplatin, the trans-isomer of cisplatin, has been discarded with recent developments. The dearth of activity of the compound was overcame by replacing and/or modifying the ammine groups with bulky ligands. This article seeks to review some of these trans-platinum compounds and their relevance in anticancer chemotherapy.
Sunday
3753925 - Dynamics and oligomerization state of the alternatively spliced, disordered region of the MEF2D transcription factor modulate cell differentiation | Poster Board #2603
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The MEF2 transcription factors play crucial roles in muscle cell myogenesis and morphogenesis.
Bioinformatical analysis indicates that the alternatively spliced β-domain of MEF2D does not fold into a well-defined structure and likely remains conformationally heterogeneous upon interactions. To study the role of protein dynamics in the biological function, a series of dynamical variants of the 37 AA of MEF2D β-domain were designed, affecting the conformational transition upon binding.
The dynamics of the free peptides has been studied using 15N relaxation measurements (T1, T2, heteronuclear NOE) from which reduced spectral density functions were calculated. Random Coil Indexes (RCI) were determined from the chemical shifts (CA, CB, N) to derive model-free order parameters. Diffusion NMR-spectroscopy (DOSY) showed the differences between the degree of structural compactness and aggregation properties. Experimental NMR parameters were comparable to those obtained from 3x100 ns long molecular dynamics simulations (MD). Order parameters of the backbone amide bond vectors, the propensity of transient secondary structure elements, radius of gyration and other descriptors of disorder were calculated. Detailed analysis of the dynamical and structural properties of the peptides will give insights into the altered interaction patterns of the MEF2D β-domain with altered biological outcomes.
Left: NMR s<i>tructure of MEF2-DNA complex (PDB:1c7u), MEF2D is displayed by green and red cartoon, scattered lines indicate regions with missing electron density. The localization of the β-domain is represented by black circles. Right: comparing the results of diffusion NMR-experiments (37AA peptides).</i>

Left: NMR structure of MEF2-DNA complex (PDB:1c7u), MEF2D is displayed by green and red cartoon, scattered lines indicate regions with missing electron density. The localization of the β-domain is represented by black circles. Right: comparing the results of diffusion NMR-experiments (37AA peptides).


Sunday
3753949 - Mechanism and activity of an L-threonine transaldolase enables implementation in a C-C bond forming biocatalytic cascade | Poster Board #3002
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
L-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5’-phosphate (PLP) dependent enzymes. In this work, we describe the catalytic mechanism of ObiH, an lTTA present in the biosynthesis of the β-lactone natural product obafluorin. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of L-threonine (Thr) to form a remarkably persistent carbanion-like glycyl quinonoid intermediate, with a half-life of ~ 3 h. Protonation of this intermediate is kinetically disfavoured, enabling on-cycle reactivity with aldehydes to form β-hydroxy-α-amino acids. Access to the native electrophile class for ObiH, α-aryl aldehydes, is precluded due to the innate reactivity of these molecules. We therefore developed a chemoenzymatic platform to generate α-aryl aldehydes utilizing styrene oxide isomerase (SOI) and styrene oxide analogs. The two-enzyme cascade was successful at generating and capturing unstable α-aryl aldehyde intermediates under aqueous conditions. Co-expression of both enzymes in E. coli yields a whole cell biocatalyst capable of synthesizing a variety of stereopure β-hydroxy-α-amino acids. We used isotopically labelled substrates to probe the mechanism of SOI, which we show to catalyze a concerted isomerization featuring a stereospecific 1,2-hydride shift. The mechanistic investigation of ObiH and SOI provide a thorough analysis of the reactivity observed through this two-enzyme cascade and demonstrate that α-aryl aldehyde intermediates are useful synthons in C-C bond forming cascades.

Sunday
3754242 - Modulation of cell surface glycans and resulting effects on calreticulin binding and phagocytosis | Poster Board #2705
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Logan Zettle, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Calreticulin (CRT), an Endoplasmic Reticulum (ER) resident protein, can promote recognition and phagocytosis of cancer cells by macrophages when present on the surface of these cells. This surface localization may be due to translocation from the ER, or exogenously bound CRT secreted from macrophages. Recently, it has been discovered that removal of terminal N-acetylneuraminic acid (Neu5Ac) on malignancies reveals a cryptic tri-antennary/multivalent (Tri/Mii) glycan which binds secreted CRT, increasing phagocytosis. In this work, we utilize sialyltransferase inhibitors to modulate surface Neu5Ac levels, leading to increased Tri/Mii moiety exposure, exogenous CRT binding, and phagocytosis. Tests were conducted using a range of hematopoietic cell lines, and pro-inflammatory macrophage-like cells were differentiated from the THP-1 monocyte cell line using phorbol 12-myristate 13-acetate and lipopolysaccharide. Recombinant CRT was fluorescently labelled and used to quantify surface binding. Surface glycan modulation and quantification of phagocytosis were conducted using flow cytometry. Surface CRT binding was successfully increased, and a resultant increase in phagocytosis was observed.
Sunday
3754281 - Formation of metallothionein-3 Cu(I)4-thiolate cluster proceeds through a long-lived, Cu-coupled, and oxygen-inert disulfide radical anion | Poster Board #3013
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Mammalian metallothioneins (MTs) are small (6-7 kDa), cysteine-rich proteins that bind with high affinity d10 essential metals, Zn(II) and Cu(I), and upon exposure, non-essential toxic metals like Cd(II). Apo MTs are unstructured, but Zn(II)-binding drives MT folding into a 3D topology characterized by two domains encompassing metal-thiolate clusters: a Zn(II)3CysS9 cluster in the N-terminal β-domain and a Zn(II)4CysS11 cluster in the C-terminal α-domain, with each Zn(II) tetrahedrally bound to a conserved array of 20 cysteine residues acting as terminal and/or bridging ligands. Contrarily, copper binding to MTs result in the formation of digonally/trigonally bound Cu(I) in Cu(I)4/Cu(I)6-thiolate clusters in each domain, with the β-domain having a higher Cu(I)-binding stability. Interestingly, studies on the differences in the preferential binding of copper to the human MT isoforms have revealed MT-3 to have the highest copper-thionein character, with its Cu(I)4-thiolate cluster formed in the N-terminal domain found to be unusually inert to oxidation in the presence of molecular oxygen.

Using a combination of biophysical and spectroscopic approaches, we investigated the chemistry and pathway of assembly for the formation of the Cu(I)4-thiolate cluster in the final Cu(I)4Zn(II)4MT-3 product upon the reaction of Zn7MT-3 with Cu(II). MT-3 thiolate residues can reduce Cu(II) to Cu(I) in a concerted electron transfer process concomitant with the formation of intramolecular disulfide bonds. Stopped-flow electronic absorption spectroscopy revealed rapid formation and consumption of transient disulfide radical anion (DRA) species centered at 430-450 nm, which have lifetimes in the seconds regime. By rapidly freeze quenching the reaction intermediates, the DRA nature of the sulfur-centered species was confirmed by both Raman and Electron Paramagnetic Resonance (EPR) spectroscopies, with the EPR simulations revealing that the DRA intermediates are coupled to Cu(I). Overall, we were able to dissect and provide evidence that the pathway of assembly and formation of this unique Cu(I)-thiolate MT-3 cluster with short Cu-Cu distances (<2.8 Å), as verified by low temperature luminescence spectroscopy, proceeds through the formation of long-lived, Cu-coupled, and oxygen-stable disulfide radical anion intermediates.

Sunday
3754303 - Investigating the effects of copper and copper chelating compounds on Caenorhabditis elegans with pan-neuronal amyloid beta 1-42 expression | Poster Board #2605
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The leading hypothesis for Alzheimer’s Disease (AD) is the aggregation of amyloid beta plaques in the brain, but research has yet to definitively prove the role of the amyloid peptide in the pathology of the disease. Our work focuses on investigating the interaction of amyloid beta peptides with copper to study the effects on neurotoxicity and amyloid-beta aggregation in vivo. The interaction between amyloid beta and copper results in the production of reactive oxygen species. To combat this, we use copper chelating compounds to prevent the interaction between copper and amyloid beta. The effects of copper and the various copper chelate compounds on the mobility and lifespan of Caenorhabditis elegans (C. elegans) with Pan-neuronal amyloid beta1-42 expression have been studied in comparison to a control group. Copper chelation reduces the amount of reactive oxygen species produced in the brains of the C. elegans. There is also a positive relationship between a mixture of copper and the copper chelator, beta-alanine, and increased lifespan in the experimental C. elegans. This suggests that the increased lifespan on the C. elegans occurs because beta alanine binds to copper (and potentially other metals), reducing the overall formation of reactive oxygen species in neuronal.
Sunday
3754329 - 13C electron nuclear double resonance spectroscopy reveals the structure of the active site enzyme-substrate complex of an active, N-linked glycosylated lipoxygenase | Poster Board #2808
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Lipoxygenases (LOX) from pathogenic fungi belong to the LOX family of enzymes, that catalyze C−H activation of polyunsaturated fatty acids to form a diverse set of cell-signaling hydroperoxides. The fungus Magnaporthe oryzae, is the most devastating plant pathogen known so far, secretes its LOX named MoLOX at the site of infection along with lipases and has been considered one of the prime suspect causing rice blast disease. Thus development of inhibitors could help to establish a pathogenic role of MoLOX in rice blast disease and potentially provide a successful intervention for it. MoLOX are glycoproteins decorated with N-linked glycans, but the impact of N-linked glycans on the structure and function of these enzymes remains largely unknown as glycans inherently prevents crystal packing and thus it lacks high-resolution structural information on the active enzyme-substrate (ES) complexes. We have previously applied 13C electron nuclear double resonance (ENDOR) spectroscopy to capture a high-resolution, 3-D structural information of the elusive ES complex of the prototypical plant lipoxygenase from soybean (SLO) with substrate linoleic acid, LA. We extend our studies and here reveal the geometrics of the active site ES structure for the native, N-linked glycosylated form of MoLOX using 13C ENDOR spectroscopy. We have further monitored changes in the ES ground state structure of the native MoLOX:LA complex after clipping glycans using ENDOR. ENDOR results combined with kinetics data report on the effects of glycan processing on the ground-state and tunneling-ready states of the ES complex. The results also provide unique insights into functional differences among LOX highlighting the robustness of the combined application of ENDOR and molecular dynamics simulation (MD) to characterize the active site ES structures of lipoxygenases that are often elusive to X-ray crystallography. The structural information gleaned from the current work is expected to aid in guiding future in silico screening efforts and rational inhibitor design of MoLOX.
Sunday
3754403 - Understanding the molecular mechanism of how histidine kinase, NahK, influences virulence in pseudomonas aeruginosa. | Poster Board #2609
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. The most at-risk patients are those on ventilators, a problem intensified by increased ventilator use during the Sars-Cov-2 pandemic. Because prevalent P. aeruginosa strains are becoming more multidrug resistant, it is essential that we understand the underlying molecular mechanisms of these infections.
The major molecular network that controls Pseudomonas biofilm formation is the GacS Multi-Kinase Network (MKN). This network is responsible for the switch between a motile, acute infection and a biofilm, chronic infection. The regulation of this switch is critical as the bacteria can seamlessly transition between each state in vivo to cause infection. However, the details driving this switch are unknown. This network is implicated in many essential processes including quorum-sensing, antibiotic resistance, metabolism, and virulence.
My goal is to understand the distinct function of histidine kinase, NahK, in the GacS MKN. My preliminary data shows that a P. aeruginosa nahK deletion strain significantly increases exotoxin and quinolone production, suggesting NahK has an important regulatory role in controlling expression of virulence factors that was previously unknown. In this project, I plan to investigate the signaling network downstream NahK to understand how this kinase affects virulence through biochemical and genetic assays. I also plan to study NahK’s interactome through in vivo and in vitro assays as there are currently no other known binding partners for this protein and recent data about other MKN kinases show that several heterodimerize and lead to changes in gene expression.

Sunday
3754468 - Strategies for next-generation flavivirus vaccine development | Poster Board #2613
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Dengue virus (DV) is the most common mosquito-borne virus, with 4 billion people living at risk for infection by its four serotypes (DV1-4). Infection by DV and its related flavivirus Zika virus (ZV) may clinically manifest as mild flu-like symptoms and arthralgia. Severe DV cases can progress into hemorrhagic fever and shock syndrome, while severe complications of ZV infection include Guillain-Barre syndrome and neonatal microcephaly. Severe dengue is associated with antibody-dependent enhancement (ADE) of infection, a potentially fatal phenomenon. In response to a primary DV infection, the immune system produces antibodies that can bind and neutralize that serotype to resolve infection. However, during a subsequent infection by a homologous DV serotype or ZV, antibodies are re-elicited but may be non- or weakly-neutralizing; thus, instead of eliminating infection, these antibodies promote uptake of active virus into Fcγ receptor-expressing immune cells and increase viremia and inflammation. Currently, there is a need for vaccines for DV and ZV that do not carry the risk of inducing ADE. We determined E glycoprotein as a favorable candidate for immunogen design. Neutralizing, protective antibodies have shown to target domain III (DIII) of E glycoprotein, which is critical for viral entry and cell attachment. However, non-neutralizing, ADE-inducing antibodies were also identified to bind to other DIII motifs. This proposal will investigate structure-based methods to display DIII immunogen multivalently and to increase the immunodominance of heterologous favorable epitopes found on the lateral ridge (LR) of DIII, while masking unfavorable ones on, to induce broad humoral protection and mitigate severity of disease. We previously demonstrated strong immunoprotecting potential of a homotypic ZV immunization strategy, which involved the multivalent display of a LR-focused DIII mutant onto nanoparticles. We will study the effectiveness of cocktail and mosaic nanoparticles as heterotypic immunization strategies against ZV and DV and characterize underlying humoral responses to the immunizations in mice. Sera of vaccinated mice will undergo neutralization assays against DV-ZV. B cells will be evaluated for sequence and lineage diversity of the resulting antibody repertoire. These studies will further our understanding of the adaptive immune response to flavivirus infections and may provide a framework for developing novel vaccines and antibody-based therapeutics.
Sunday
3754483 - Development of a cell-based AlphaLISA assay and utilization in a high-throughput screen for small molecule proteasome enhancers | Poster Board #2905
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
As we age, proteasome function decreases, resulting in the accumulation of intrinsically disordered proteins (IDPs), which has been directly linked to many neurodegenerative diseases. This highlights the need to develop methods to prevent or halt the dysregulation or accumulation of disordered proteins. Enhancement of the proteolytic activity of the proteasome offers a promising method to prevent IDP accumulation. However, methods to evaluate and probe this activity remain limited. Furthermore, the available activity assays are not easily modified to meet high through-put screening demands. To meet the demands of a high-throughput screen, we describe the development of a cell-based AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) designed to measure the rate of proteolytic degradation of a well-known IDP, ornithine decarboxylase (ODC), that is tagged with a green fluorescent protein (GFPSpark) on its N-terminal. GFPspark folds much quicker than other GFP counterparts while maintaining the stable characteristic β-barrels. Its high degree of stability proves to be a challenge for even the 19S cap, of the 26S proteasome complex, to unfold. ODC, on the other hand, is a homodimeric enzyme that plays a key role in the biosynthesis of polyamines. In its monomeric form, ODC is inactive, intrinsically disordered, and can be degraded by the 20S proteasome. The opposing structural characteristics of GFPspark and ODC, make their conjugated protein combination, an ideal substrate for the evaluation of 20S proteasome activation by small molecules. The use of the cell-based AlphaLISA enabled us to screen the NIH Clinical Library and the Prestwick library, and we identified Erlotinib as a new 20S proteasome enhancer.
Sunday
3754580 - In vitro determination of zinc coordination complex of Acinetobacter baumannii Zur | Poster Board #2615
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Bacterial proteomes consist of 30% metalloproteins, which require their cognate metal for proper structure or function. To avoid the binding of higher affinity metals to all metalloproteins, cells precisely regulate and maintain the bioavailable concentration of each metal in an inverse order of the stability of the metal complex, known as the Irving Williams series (Mn2+2+2+2+<2+>Zn2+). To maintain metal homeostasis, bacterial transcriptional metal sensors regulate bioavailable metal concentrations. Zinc uptake regulator (Zur) is a ferric uptake regulator (Fur)- family metalloregulator that represses transcription of genes by dimeric binding of DNA. Zur’s DNA affinity increases as zinc binds to a regulatory site. In response to the fluctuation of zinc availability, Zur regulates the genes encoding proteins that mediate metal uptake, export, and allocation. We propose that the in vitro range that Zur responds to would be an initial reference to the bioavailable zinc in the cellular environment. Here, we have elucidated the range of free Zn concentration over which the sensor responds in vitro using fluorescence anisotropy. Additionally, we have structurally determined that the regulatory zinc-binding residues in Acinetobacter baumannii Zur are C100S, H89A, H107A, and E122A using XAS(X-ray absorption spectroscopy), Co spectra, and IVT(In vitro transcription). Thus, we propose the coordination chemistry of the regulatory site of AbZur as well as the Zn bioavailable sensing range in A. baumannii.
Sunday
3754600 - Inhibition of STING signaling by harnessing its autoinhibitory mechanism | Poster Board #2617
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
STING is a receptor central to an innate immune pathway that detects cytosolic double-stranded DNA and mediates antiviral and anticancer immunity. STING activation hinges on its nanomolar affinity binding to a small-molecule second messenger, 2’,3’-cGAMP, making this pathway uniquely chemically tractable amongst innate immune mechanisms. For this reason, agonists of STING are in intense clinical development as a means of stimulating anticancer immunity at the innate immune level, but few inhibitors of STING are known. However, in a broad range of tissue-specific and systemic autoimmune diseases, failure to regulate self-DNA leads to mis-activation of the STING pathway, resulting in unwanted inflammation. It is thus of therapeutic interest to investigate how inhibition of the STING pathway is relevant in broad range of disease contexts. We hypothesize that examining the molecular mechanism of STING activation can inform new approaches to inhibit this innate immune pathway. We previously described a model of STING activation where STING is basally autoinhibited. We showed that autoinhibition is relieved by 2’,3’-cGAMP binding, and can be exploited to achieve STING inhibition. Here, we elaborate on this model and show that peptides can be used to mimic autoinhibition in the native cellular context. Furthermore, we developed assays to quantify peptide binding to STING, and established residue-affinity relationships. This work paves the way for the development of molecular tools to inhibit STING signaling to study its functional consequences in various disease outcomes.
Sunday
3754638 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3754641 - Aminopyrrolnirtin oxygenase (PrnD); a study of the final O2-dependent enzyme involved in pyrrolnitrin biosynthesis in Burkhorderia ambifaria | Poster Board #2700
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Pyrrolnitrin is an antimicrobial and therapeutic derivative of tryptophan used in agricultural, pharmaceutical, and industrial settings. The biosynthetic pathway from tryptophan requires four enzymes encoded by the prnABCD cassette. These enzymes have been initially characterized from Pseudomonas fluorescens, however little has been done with the pyrrolnitrin biosynthetic pathway from Burkholderia ambifaria. Here we report our focused efforts on the expression, purification, characterization, and catalytic reactivity of the last enzyme in pyrrolnitrin biosynthesis, PrnD. PrnD is an aminopyrrolnitrin oxidase, which contains a 2-his-1-carboxylate, non-heme iron center and a Rieske-type diiron center. Preliminary characterization data of B. ambifaria PrnD is compared with data reported for P. fluorescens PrnD. The turn-over efficiency of this system is studied using a newly developed NADPH-dependent activity assay for catalytic oxidation of aminopyrrolnitrin. The specific focus of this work is focused on the biophysical characterization of enzyme/substrate binding dynamics, where this data is compared to other non-heme iron oxygenases.
Sunday
3754684 - Probing the structure of the caspase-cleaved prostate apoptosis response-4 (cl-Par-4) tumor suppressor | Poster Board #2903
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein with significant intrinsic disorder character. Par-4 is unique in the sense that it can selectively induce apoptosis in cancer cells. Down-regulation of this protein has been reported in a myriad of cancers whereas up-regulation is associated with several neurodegenerative disorders. Caspase-dependent intracellular cleavage of Par-4 is essential to produce the functionally active fragment, cl-Par-4 (caspase-cleaved Par-4) which enters the nucleus and inhibits NF-kB-mediated cell survival pathways. Detailed structural information of cl-Par-4 will be helpful in designing a promising strategy to re-establish normal Par-4 activity inside cells. Here, we have employed circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) to analyze the conditions under which cl-Par-4 will be suitable for structural analysis via NMR spectroscopy and X-ray crystallography. We have compared size, secondary structure and solubility of cl-Par-4 as a function of ionic and pH environment. We have also assessed the secondary structure of the D313K point mutant of cl-Par-4. These findings will be helpful in understanding function and for solving the structure of cl-Par-4.
Sunday
3754811 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3754830 - Structural and functional characterization of lysyl oxidase-like 4 | Poster Board #2706
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Lysyl oxidases are a family of enzymes that contains lysyl oxidase (LOX) and lysyl oxidase-like isoenzymes 1-4 (LOXL1-4). All of the enzymes in the family are known to share a conserved C-terminal, while differing in the primary sequence of the N-terminal domain. The primary role of the lysyl oxidase enzymes is in the remodeling of the extracellular matrix (ECM) via the oxidation of specific lysine residues to aldehydes, thereby catalyzing the crosslinking of collagen and elastin. This reforming of the ECM has been deemed the mechanism through which LOXL-4 may promote cancer metastasis. The work for this project is well-underway as the LOXL-4 DNA has been verified through DNA sequencing, and the LOXL-4 protein has been overexpressed and visualized via SDS-PAGE. The main goal of this research is to obtain a crystal structure for LOXL-4. The crystal structure will provide insight into the structure of the protein, which has yet to be elucidated, and how it folds. The structure will also help to determine active sites in the molecule that are crucial for designing inhibitors. Even though the COVID-19 pandemic has delayed the investigation of this enzyme, protein crystals for LOXL-4 has been successfully obtained. Now that conditions for crystal formation have been identified, the next step is to optimize crystal growth using 96-well sitting drop plates and 24-well plates using both a hanging drop and sitting drop method. Additionally, assays will be conducted to determine activity, copper incorporation, and BAPN inhibition. Lastly, the kinetic constants km and kcat will be determined, along with kI for various inhibitors designed by the Solano research group.
Sunday
3754834 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3754883 - Analysis of metal binding in blue fluorescent protein using polarized resonance synchronous spectroscopy | Poster Board #2536
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Mutants of green fluorescent protein (GFP) have been used as biosensors for many purposes, including the monitoring of temperature, pH, and metal binding capabilities. While GFP does not show appreciable metal binding abilities, mutants of blue fluorescent protein (BFP) can bind copper, zinc, and possibly other metal ions effectively. Protein binding of these ions is typically monitored using fluorescence techniques, as the binding of metal ions to BFP mutants appreciably changes the observed fluorescence. In this work, further fluorescence characterization of metal binding by BFP is carried out using the recently developed polarized resonance synchronous spectroscopy (PRS2) technique combined with traditional fluorescence methods. PRS2 allows for the separation of the fluorophore on-resonance fluorescence (ORF), which is the photon emission at the same wavelength as the excitation photon, and light scattering. This technique could be further applied to monitor the movement of metal ions in living cells.
Sunday
3754922 - Synthetic characterization of brucebactin and recognition by its periplasmic partner FatB | Poster Board #2701
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Brucella spp. is a pathogen that infects many farm animals, causing abortion in these animals. Humans can acquire this infection zoonotically by coming in contact with infected farm animals or consuming/handling milk/meat from infected animals. Human-to-human transmission is rare, so controlling human brucellosis requires preventing the infection in its primary hosts. Like many other pathogens, Brucella requires iron for its survival and virulence and in the production of its siderophores. Siderophores are small organic molecules that acquire iron for these microbes. The siderophore produced by Brucella is Brucebactin (BB). However, the exact chemical nature remains uncharacterized. I have synthesized a di-catechol siderophore based on literature evidence, which is believed to be BB. The synthesis involved a mixture of 2,3-dihydroxybenzoic acid (DHBA), potassium carbonate, and benzyl bromide dissolved in a solution of acetone and refluxed overnight. Then the residue underwent saponification at the carboxylic acid via dissolution in methanol and sodium hydroxide before being refluxed for three hours. The residue was dissolved in water and acidified with hydrochloric acid. The precipitate was then isolated and dissolved in dichloromethane, spermidine, and tetramethylammonium tetrafluoroborate (TBTU). The product obtained (BB) was purified via column chromatography. Structure elucidation testing, which included Proton and Carbon NMR, Mass spectrometry, and Infrared spectroscopy, was collected to verify each intermediate product's correct structure in the synthesis. Using this newly characterized BB, the interaction with its cognate periplasmic protein FatB will be verified through Isothermal titration calorimetry (ITC). The recognition of BB by its periplasmic partner will allow future research to focus on targeting this bacterium’s active transport systems to facilitate drug entry via a peptide linker using a trojan horse approach.
Sunday
3754930 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3754945 - Structure-guided photoaffinity ubiquitin probes give insight into deubiquitinase binding sites | Poster Board #2714
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Ubiquitin (Ub) is a post-translational modifier crucial to eukaryotic biology. The Ub system relays an intricate control over cellular processes through its attachment and detachment on substrate protein lysine residue, giving rise to a “ubiquitin code”. Deregulation in this code has severe consequences, primarily in disease pathogenesis. Proteolytic deubiquitinating enzymes (Dubs) render this PTM reversible by removing or “trimming” Ub off substrate protein. In turn, this interplay between the attachment and detachment machineries establishes a well-defined niche of ubiquitinated protein to relay critical cellular information. Ub modification, unlike other PTMs, is multivalent. Subsequent additions of one Ub moiety to 1 of 8 amines (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys68) on another confers the assembly of polyUb chains with well-defined linkages, each relaying a vastly different function. Dubs must recognize this multivalency by accommodating several topologies of Ub chains. Here we show that genetically encoded photophore Bpa in the Thr-9 position serves as a pan-Dub probe that gives insight into Ub binding sites. We demonstrate that genetic code expansion (GCE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) gives structural insight into the Ub-Dub interaction mode.
Sunday
3754949 - Molecular and bioinformatic studies of the Neisseria meningitidis serogroup W capsule polymerase | Poster Board #2718
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Neisseria meningitidis serogroup W (NmW) is one of the leading causes of bacterial meningitis. The NmW capsule polymerase enzyme creates the capsular polysaccharide of this serogroup. This 120 kDa enzyme has three domains: an N-terminal galactosyltransferase (GT), an intervening sequence, and a C-terminal sialyltransferase (ST) domain. There is no known three-dimensional structure of the protein. The overall goal is to understand the structure-function relationship of the NmW capsule polymerase. The goals of this project are to (1) Polymerase Chain Reaction (PCR) to amplify the gene sequence for a protein expression optimized version of the NmW capsule polymerase and ligate into a protein expression plasmid and (2) to create a computational model of the sialyltransferase domain. For the first goal, PCR primers were designed to amplify the DNA of the NmW enzyme and introduce restriction enzyme (RE) sites BamHI and XhoI for future ligation. Next, plasmid DNA was obtained by mini-prep from overnight growth of non-optimized NmW enzyme. Characterization of the NmW plasmid DNA (four samples) were performed by absorbance (Nanovue). An enzyme digest was done to remove the previous DNA insert from the previous expression. For protein modeling studies, the amino acid sequence of the NmW capsule polymerase was entered into ProtParam to aid in identification of the sialyltransferase domain sequence. This domain’s sequence was then input into I-TASSER. Results indicate successful amplification of the PCR insert and the non-optimized plasmid was obtained at high concentration (best sample = 212.5ng/uL) and purity (A260/A280 =1.937). However, RE digest results were inconclusive. One reliable structural model was obtained (C score = -0.71). In future work, we will repeat the RE digest and use model to help in understanding the active site.
Sunday
3755033 - Investigating the role of naturally occurring modifications in U2 snRNA under oxidative stress | Poster Board #2703
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Oxidative stress, which results from the overproduction of free radicals such as reactive oxygen species (ROS), has been associated with many degenerative diseases. These free radicals can damage cellular components including DNA, RNA and proteins. Although oxidative damage to DNA and its consequences have been well documented, damage to RNA has been less studied. Many post-transcriptional modifications of RNA have been reported to play an essential role in snRNA biogenesis and pre-mRNA splicing. Moreover, evidence suggest that the human spliceosomal machinery may be stabilized by the extensive modifications in U2 snRNA. In humans, this oligomer contains methylated guanosine (m6G), 2'-O-methylated sugars, and pseudouridine. The aim of this study is to investigate the role of naturally occurring modifications in U2 snRNA in maintaining its structure and function in an oxidative environment. For this purpose, twelve modified nineteen nucleotide RNA oligomers representing the branchpoint recognition site of U2 snRNA will be synthesized with incorporation of modifications at positions 28 through 46. These chemically synthesized oligonucleotides will be analyzed using UV-Vis, HPLC, MALDI and CD to determine the effect of site-specific modification on structural features of U2 snRNA. This information will be foundational in determining the correlation between structural characteristic of the RNA and the damage products obtained through site specific radical generation in related substrates. Findings of this research will provide tools for the study of oxidative damage to RNA and key roles of naturally occurring mutations in the structural stability of the spliceosomal complex under oxidative stress. It will further shed light on the underlying mechanisms behind the contributions of RNA oxidative damage to degenerative diseases.
Sunday
3755100 - Excision of oxidatively damaged bases in G-quadruplexes by the DNA glycosylases NEIL1 and NEIL3 | Poster Board #2707
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Environmental toxins, endogenous metabolic products, and ionizing radiation can directly modify DNA or indirectly damage DNA through the generation of reactive oxygen and nitrogen species (RONS). This DNA damage arises at a rate of thousands per day compromising the integrity of the genome. The endonuclease VIII-like (NEIL) family of DNA repair glycosylases initiate repair of the damage produced by RONS. They exhibit particularly high activity toward the removal of hyperoxidized products of guanine, such as the guanidino- and spiriminodi-hydantoins (Gh and Sp), which mediate G-to-T and G-to-C transversion mutations and serve as replication blocks. The NEIL family is also capable of excising these lesions from a variety of DNA contexts, such as duplex, single-stranded, bubble, and bulge DNA; notably, NEIL1 and NEIL3 both have the unique ability to excise Gh and Sp from G-quadruplex (G4) DNA. Due to the location of these G4 DNA structures in various oncogene and repair enzyme promoter regions, understanding how NEIL processes damaged bases in a G4 context can provide insight into the unique roles of NEIL beyond genome maintenance. We have conducted a thorough kinetic and binding affinity analysis of the repair of Gh lesions from different G4 sequences by NEIL1 and NEIL3. Specifically, under single turnover conditions, the removal of Gh by NEIL was examined in G4 sequences from the VEGF, KRAS, and RAD17 promoter regions. Interestingly, the observed production curves for NEIL-catalyzed base removal are biphasic, indicating that the substrate is processed at two distinct rates, and this is highly dependent on the sequence and location of Gh within the G4. Our most striking observation indicates that a significant amount of substrate remains unprocessed despite enzyme being present in excess. This suggests that the G4 structure makes an impact on the overall extent of Gh removal. Binding studies suggest that NEIL may be binding in an unproductive manner that does not support Gh excision. Our results showcase the interaction of NEIL with G4s and provide insight into a possible dynamic relationship between the NEIL family of enzymes, DNA repair, and gene regulation.
Sunday
3755112 - Development and modification of protein material for general synthetic applicability | Poster Board #2709
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Enzymes are powerful catalysts that perform reactions with high selectivity and turnover while being relatively green and non-toxic. However, despite advances of enzyme reactivity via rational design and directed evolution, solvent compatibility remains an issue for general applicability. Protein-based materials can potentially be used to circumvent this issue. Like metal organic frameworks and covalent organic frameworks, some protein-based materials can maintain their innate catalytic activity even under organic solvent conditions. The functional groups displayed within the pores of these protein-based materials provide unique chemical environments that can allow for modification and catalytic properties not accessible to other materials. These materials will be modified and explored for cascade enzymatic and synthetic catalytic reactions.
Sunday
3755114 - Insight into the regulation of ADP-glucose pyrophosphorylase: Importance of a homologous serine and threonine in the allosteric binding site | Poster Board #2719
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) is directly responsible for the production of ADP-glucose, the fundamental molecule in the production of starch in plants and glycogen in bacteria. There are several model organisms that can be used to study and explore this enzyme, all of which are regulated allosterically by a variety of sugar phosphates that are specific to each organism. In Melainabacteria, one of the model organisms chosen for our study, these activators are fructose 6-phosphate (Fru6P), glucose 6-phosphate (Glc6P), and mannose 6-phosphate (M6P). Several different residues have been identified as impacting the allosteric regulation of ADP-Glc PPase. One of the residues studied was serine 72 in ADP-Glc PPase from Agrobacterium tumefaciens. This residue when mutated to other residues like alanine and glutamic acid, showed a decrease in activity and affinity for the activator Fru6P. Here we investigate the homologous residues Thr79 in E. coli and Ser65 in Melainabacteria. We mutated the Thr79 to a variety of different residues that included alanine and serine, these changes caused a decrease in overall enzymatic activity but more importantly, it had an effect on the binding affinity of the activator to the allosteric site. It is believed that the cause of this effect was because the residue is found in the allosteric binding site of the activator, fructose 1,6-bisphosphate (FBP), in E.coli. In Melainabacteria the homologous residue to Thr79 and Ser72 is Ser65. Therefore, to study this residue in Melainabacteria we conducted a point mutation to switch the Ser65 residue to an alanine to investigate how a change in amino acid characteristics would change the overall enzymatic activity. Since the residue in Melainabacteria is located in the allosteric site where its corresponding activators bind, we predict that this residue is also responsible for the binding of these activators. If this residue does indeed have an impact on the regulation of ADP-Glc PPase in Melainabacteria it can help us understand how the enzyme is regulated. In conclusion, we hope to lay the groundwork for characterizing the regulation ADP-Glc PPase. This knowledge could be used for biotechnological purposes, such as increasing the production of starch in plants and biofuels.
Sunday
3755230 - Patient derived glioma stem cell modulation for improved therapeutic outcome | Poster Board #2711
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Glioblastoma Multiforme (GBM) falls under the category of the most lethal malignant primary brain tumors owing to their heterogeneity and self-renewal capacity. Modulation of the glioma stem cells (GSCs) using chemotherapeutics to specifically target these heterogeneous stem cell populations resulting in complete elimination of the GSCs still remains a major challenge. The GSC population is resistant to all the available conventional therapeutic strategies thereby making GBM highly recurrent and extremely aggressive in nature. This demands an urgent need to develop therapeutic strategies against the highly resistant GSCs. The patient-derived glioma stem cells isolated from surgically resected tumor tissues were grown in culture for determining the characteristic features responsible for increased stemness characteristics in this cell population. Our results so far documented that the patient-derived GSCs of varied backgrounds utilize pathways for their growth and survival that might be different from that of the bulk tumor. Preliminary studies show that inhibition of such pathways in GSCs can potentially lead to a change in their characteristic features. Thus, we embark on a journey to find a promissory way that can make the GSCs vulnerable. In this presentation, we will be presenting our primary findings in vitro and in vivo using the various formulations that can show metabolic inhibition to set a stage for understanding and analyzing how attacking GSCs can affect the GBM tumor progression.
Sunday
3755241 - Characterization of a heme-responsive NosP signaling pathway in bacteria | Poster Board #2715
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Bacterial biofilms are surface-attached communities of bacteria formed within a self-secreted exopolysaccharide matrix. Formation of biofilms is a survival strategy adopted by bacteria, that enables them to take up nutrients in a low nutrient environment and provides a safe niche where the bacteria are protected from fluctuations in temperature and pH. It also protects the bacteria from host immune responses and harsh chemical treatments which renders them resistant to conventional antibiotics. This makes biofilms a growing threat to human health as most hospital acquired bacterial infections involve the formation of biofilms on stents, catheters, and medical implants. Biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa, upon infecting the lungs of patients with cystic fibrosis, is also the leading cause of death in these patients. Therefore, it is essential that investigation be carried out about the molecular mechanisms underlying the signaling pathways involved in biofilm regulation, in order to develop more efficient strategies to treat and eradicate biofilm associated bacterial infections.
NosP, a novel Nitric Oxide Sensing Protein discovered in our lab, is a hemoprotein that has been demonstrated to regulate biofilms through two-component signal transduction networks. The first step involved in the NosP mediated signaling pathway is the sensing of the diatomic gas molecule nitric oxide (NO), which induces biofilm dispersal through quorum sensing, or histidine-containing phosphotransfer protein (specifically, HptB) signaling involving a multikinase network, or through modulation of the intracellular concentrations of cyclic diguanylate monophosphate (cyclic-di-GMP), a secondary messenger molecule that has been implicated in biofilm regulation. These signaling pathways demonstrate the role of NosP as a heme-based gas sensor. However, recent studies with the NosP in Vibrio cholerae (Vc0130 or VcCdpA) have shown that the N-terminal NosP domain binds heme flexibly, which regulates the phosphodiesterase activity of its C-terminal domain. This suggests that NosP may also function as a heme-responsive sensor protein. The aim of my project is to characterize this recently discovered function of NosP and to investigate the role of heme in the NosP mediated signaling pathways in bacteria.

Sunday
3755358 - Multivalent, PSMA-targeting megamolecules as radiopharmaceuticals for prostate cancer | Poster Board #2800
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
In this work, a new class of antibody mimics was evaluated; specifically, the ‘megamolecule’ platform developed in our laboratory, where synthesized ligands react with recombinantly expressed fusion proteins to create multi-component, complex architectures. With this platform, a protein complex containing small molecule inhibitor ligands that bind to the cell surface protein prostate-specific membrane antigen (PSMA), which is commonly overexpressed on prostate cancer cells, was designed. The protein foundation of the platform allows it to span the distance required (~100 nm) to react with multiple PSMA proteins on the cell surface. Subsequently, the multivalency of the complex increases the binding affinity and selectivity to PSMA and by extension, prostate cancer cells. These structures offer a completely novel opportunity to boost the efficacy of immunotherapies and targeted therapeutics and diagnostics.
Sunday
3755359 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3755435 - Mechanical regulation of extravasation into brain parenchyma in vivo | Poster Board #3001
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Despite improvements in the treatment of primary cancers, the median survival of patients with metastatic brain lesions is approximately 7-16 months from diagnosis. About 15-25% of brain metastasis is from primary breast cancer. Therefore, understanding factors crucial to cancer cell survival in the brain following dissemination is critical for developing optimal treatments for metastatic and resistant tumors.

Human xenograft model in zebrafish with breast cancer cells is used to understand brain metastasis. The zebrafish has rapidly become a model for studying tumor behavior at different stages of the metastatic cascade due to its optical transparency for single cell imaging and homology between human and zebrafish proteins. Upon entry into the brain, cancer cells must first cross the lumenal interface of the endothelium before extravasation. Cells must then overcome this mechanical barrier and adapt to a new microenvironment. To visualize and perform real time mechanical mapping of tumor cells in vivo as they move from the lumenal spaces into the tissue and as they adapt to the brain parenchyma, polystyrene beads were introduced to cancer cells by spontaneous phagocytosis and cancer cells/beads were injected to the circulation of zebrafish. Then, broad band (7-15kHz) optical tweezer-based active microrheology was used to assess the mechanical phenotypes of cells in terms of pre-extravasation, post extravasation, and one day post-extravasation.

Mechanical mapping of human breast cancer showed cancer cells soften after extravasation into the brain parenchyma in comparison to pre-extravasation. However, cells stiffen at one day post extravasation in vivo. During extravasation process, cancer cells become more liquid-like and semi-flexible. This result indicates that breast cancer cells can tune their mechanics during extravasation process to colonize brain in vivo. Future work will focus on genetic regulation of this mechanical adaptation to investigate unique mechanical drivers of extravasation in the brain.

Sunday
3755449 - Nanodetection of a miRNA biomarker for COVID-19 patients suffering cerebrovascular problems | Poster Board #2802
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Levels of microRNAs (miRNAs) within extracellular vesicles (EVs) have been demonstrated to be useful diagnostic and prognostic biomarkers in several disease states. Testing the level of miRNA24 plays an important role in potential clinic application: it deciphers neuroimmune functions in COVID19 patients by categorizing them into post cerebrovascular (CBV) disorder COVID19 patients and non-CBV COVID19 patients.This research demonstrated a nano analysis method for trace detection of miRNA24 for COVID19 patients through Surface Enhanced Raman Spectroscopy with silver nanoparticles. With a lowest concentration of 1 attomole, the preliminary research achieved miRNA24 Raman signal enhancement of up to 600 times . Continued research to generate a standard calibration curve that may be used for quantifying the levels of miRNA24 in samples is ongoing. In this project, J.L is grateful for the intern research opportunity offered by the Academy for Advanced Research and Development (AARD).
Sunday
3755516 - Increasing the immunogenicity of influenza vaccine through covalent modification | Poster Board #2812
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Influenza has induced an epidemic every fall and four pandemics in the past century, leading to millions of infections and several thousand deaths each year. One of the primary tools in vaccine development is adjuvant design and implementation to improve efficacy of the immune response. However, progress has been undercut by a lack of mechanistic understanding of how the adjuvant stimulates the immune response. There is a need for new adjuvanted influenza vaccines that are more effective to seasonal influenza virus (IV) infections and can induce an appropriate immune response against potential pandemic IV infections and vulnerable populations. Current research in vaccine development is focused on adjuvant design and new approaches for antigen presentation (i.e mRNA, inactivated, live attenuated etc.) Our approach focuses on developing a new method for introducing an adjuvant into the vaccine via covalent attachment to the antigen itself. Previous research has demonstrated the incorporation of azido sugars and amino acids onto enveloped and non-enveloped virions for modifications to the virions via click chemistry. Utilizing this methodology, CpG-ODN adjuvants can be covalently attached to the surface of the virion via click chemistry to induce a TLR-9 response by incorporation of Ac4GalNAz on the HA and NA of Influenza A virus (A/Puerto Rico/8/1934(H1N1)). CpG-ODN 1018 has been selected as the adjuvant of due to its induction of the TLR-9 receptor in HEPLISAV-B® and the lack of FDA approved TLR-9 induction in Influenza vaccines. Analysis of IV surface glycans indicates nine N-glycosylation sites on HA and six N-glycosylation sites on NA, with NA 144-152 all bearing terminal galactosamines. The CpG-ODN will bear a cyclo-octyne warhead on the 3’ end which will undergo strain-promoted, azide-alkyne cycloaddition (SPAAC) with the azido-labeled virus (via Ac4GalNAz). We hypothesize that covalently binding CpG-1018 to IV will allow for a higher concentration of adjuvant to react with TLR-9 and will induce an enhanced response for IV vaccines that can be used in conjunction with non-covalent adjuvants to induce a robust immune response.
Sunday
3755547 - Chemical and physical biology tools to elucidate metallochaperone function in Acinetobacter baumannii | Poster Board #2820
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Bacterial infections plague hospitals around the globe and the scientific community must address the complex mechanisms that allow pathogens to persist in an infected host. Vertebrate immune systems utilize strategies to suppress bacterial growth at the host-pathogen interface. One strategy termed nutritional immunity, employs vertebrate neutrophils to starve extracellular milieu of essential nutrients. Transition metals operate as cofactors in 30% of bacterial proteomes making metal restriction a critical aspect of nutritional immunity. Many pathogenic bacteria, like Acinetobacter baumannii and Bacillus anthracis, developed adaptive responses to meet nutritional demands. Recently, COG0523 proteins have been shown to insert metal cofactors into critical enzymes, termed clients, to maintain metal homeostasis in metabolic pathways. COG0523 proteins are part of the adaptive response and are critical for bacterial survival during conditions of nutrient restriction.
A1S_0934 is a COG0523 GTPase in A. baumannii and belongs to a cluster of proteins specific to Acinetobacter bacteria. Across this cluster of proteins, sequence homology suggests similar functional characteristics. Metal competition binding assays show A1S_0934 binds divalent zinc with high affinity (KZn1 = ~1011 M-1) and contains an additional low affinity site consistent with other putative COG0523 metallochaperones. Through X-ray absorption spectroscopy, the first coordination shell of the high affinity zinc binding site confirms cysteine residues from a conserved CXCC motif contribute metal coordinating bonds. An additional nitrogenous ligand suggests coordination to an invariant histidine residue in the G3 loop of A1S_0934 essential for GTP hydrolase activity.
Additionally, yeast two-hybrid experiments revealed high-confidence interaction between A1S_0934 and MurD. MurD belongs to a family of essential ATP-dependent amino acid ligases (MurA-F) that participate in the formation of peptidoglycan. The crucial role of Mur enzymes in bacterial survival has meant they maintain a popular pathway for therapeutic targeting, resulting in successful antibiotics such as fosfomycin and vancomycin. A high-confidence interaction with MurD implicates A1S_0934 as a newly discovered member of this cell wall biosynthetic pathway. Using NMR, crosslinking approaches, and biochemical assays, we intend to validate this interaction and characterize the functional role of A1S_0934.

Sunday
3755635 - Investigating polyamidoamine dendrimers as carriers for nucleic acid nanoparticles | Poster Board #2915
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Nucleic acid nanoparticles (NANPs) are an emerging technology that is amenable to several biomedical applications. Such applications include being used as therapeutic agents, imaging, and diagnostic devices. However, because of the disruptive interactions between NANPs and cell membranes, NANPs require a carrier to enter cells. Only then will NANPs be able to successfully achieve their rationally designed functions. Here, we are investigating the use of a synthetic, biocompatible carrier - amine-terminated polyamidoamine (PAMAM) dendrimers- for the delivery of NANPs into cells. PAMAM dendrimers interact with NANPs through electrostatic interactions that take place between the positively charged surface groups on dendrimers and the negatively charged backbone of the NANPs.
Dendrimers are a lucrative carrier because they have cavities that carry cargo that are modifiable depending on the generation of dendrimer. With an increase in generation is an increase in the number of surface groups that can interact with NANPs. For example, generation 4 (G4) dendrimers have 64 surface groups and generation 5 (G5) dendrimers have 128 surface groups. In this study, the interactions between G4 and G5 dendrimers with two different NANP types (RNA rings and RNA cubes) were characterized. The NANPs differ in dimensionality which has previously shown to play a significant role in immunostimulation.
The physicochemical properties of the formed dendrimer-NANP complexes were characterized through electromobility shift assays (EMSA) to determine the optimal binding ratio. The ability of dendrimers being able to protect NANPs from enzymatic degradation was also tested by exposing the complexes to DNAse over a period of time.
Following the physicochemical characterization, the in vitro behavior of the complexes was investigated through cellular uptake studies where a fluorescently labeled NANP was used to complex with dendrimers then tracked with fluorescent microscopy.
Results showed that both the RNA rings and cubes are able to complex to each of the dendrimer generations (G4 and G5) successfully and each generation can provide protection from enzymatic degradation for a minimum of 30 minutes. Uptake assays showed that dendrimer generation influences the level of cellular uptake that takes place. NANP dimensionality is also seen to play a role in the cargo capabilities of dendrimers.

Sunday
3755643 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3755681 - Development of a calcium donor for quantitative calcium release | Poster Board #2818
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Calcium is an essential metal ion that is critical for the survival of all living organisms. For instance, during an action potential (e.g., muscle contraction), massive amounts of calcium are released from the sarcoplasmic reticulum into the cytosol. It is this change that triggers a process like heart beating, neuron signaling. Dysregulation of calcium homeostasis is also associated with a number of debilitating neurological disorders ranging from AD to Parkinson’s etc. However, the molecular underpinnings of how calcium dysregulation promotes disease progression is insufficiently understood. There are several methods to study the role of calcium at the molecular level. The first entails the application of chemical probes that can detect calcium at the cellular level. Alternatively, perturbing calcium within the cell represents a complimentary approach. However, it is currently very challenging to be able to control calcium levels in a quantifiable manner. For example, the most common method to elevate calcium levels is to add this metal ion into the cell culture media. However, this method of calcium elevation is not controllable, and the increment of calcium concentration is unknown. To overcome these limitations, we have developed a newly designed calcium donor which can elevate calcium levels in a controllable manner and demonstrate the quantification of released calcium. We expect our calcium donor can serve as a powerful tool for future calcium-related studies.
Sunday
3755687 - Determining the heat-activation and ligand gating mechanism of TRPV1 | Poster Board #2816
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Kunj Patel, Presenter; Joseph DePaolo-Boisvert, Presenter; David Minh, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Transmembrane proteins such as TRPV1 are located in the brain and the peripheral neurons are responsible for facilitating action potentials that elicit pain responses. In order to elicit pain responses, action potentials are often fired in the neurons. Research has shown that vanilloids, capsaicin, and temperature increase have been shown to open the lower pore in the TRPV1 ion channel. Here, we will discuss the implications of a replica exchange simulations on the ligand-gating and temperature-activation mechanism of TRPV1.
Sunday
3755706 - Withdrawn
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person

Sunday
3755710 - Optical control of lysophosphatidic acid and phosphatidic acid signaling | Poster Board #2801
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
This poster describes the synthesis and biological applications of photoswitchable analogs of the related glycerophospholipids lysophosphatidic acid (LPA) and phosphatidic acid (PA) for the optical control of various cellular signaling processes. The phospholipid LPA is an extracellular signaling molecule that activates a large family of G-protein-coupled receptors (LPAR). These receptors play essential roles in development and in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. PA is involved in cell growth and proliferation processes, including the mTOR and Hippo signaling pathways. We synthesized photoswitchable lysophosphatidic acid (AzoLPA) and phosphatidic acid (AzoPA) analogs by incorporating an azobenzene photoswitch into the hydrophobic lipid tails. The synthesized molecules enabled effective optical control of signaling pathways involving both phospholipids. Optical control of LPA receptor activation was demonstrated by intracellular Ca2+ release and neurite retraction under light irradiation in the presence of AzoLPA. Moreover, mTOR signaling pathway activation and Hippo signaling pathway inhibition were demonstrated with AzoPA in its light-activated, bent cis form. We believe that these photoswitchable molecules provide powerful tools for chemical biologists studying complex, dynamic lipid-mediated signaling pathways.
Sunday
3757794 - Epigenetic biomarkers of neurodegenerative disease in wastewater: Lessons from liquid biopsy of cancer | Poster Board #2311
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Background. Epigenetic changes associated with etiology and pathogenesis are currently being researched as disease biomarkers in cancer and neurodegenerative diseases (NDs). Wastewater-based epidemiology (WBE) is an untapped resource to survey for epigenetic biomarkers associated with diseases to inform public health experts at a regional level. Urine and fecal liquid biopsy sampling is currently being used to identify and monitor epigenetic biomarkers of gastric and urinary cancers but not NDs. Methods. We conducted four PRISMA systematic literature reviews in Scopus from December 2021 to January 2022 for publications reporting disease biomarkers found in wastewater (n=313), epigenetic biomarkers shed in urine or feces (n=231), epigenetic biomarkers associated with NDs (n=420), and factors or comorbidities associated with epigenetic changes in AD, PD, or ALS (n=213). Results. Studies of wastewater biomarkers, urine and fecal liquid biopsy epigenetic factors, and factor exposure epigenetics in AD, PD and ALS are primarily confined to the U.S., Europe, and southeastern Asia. The most common bioindicator organisms of disease found in wastewater are bacteria. Individual biomarkers from liquid biopsy with the highest sensitivity and specificity within multiple panels, tested primarily for methylation, include RUNX3, SOX1, IRF8, and DAPK. Of the epigenetic biomarkers found in brain or blood specimens of ND patients, global hyper or hypomethylation was reported compared to controls. Epigenetic marks associated with factors driving AD, PD, or ALS are primarily DNA methylation and histone modification. While heavy metals are quantified in wastewater to inform human risk of disease, AD is associated with air pollution (increased active marks) and lead (hypoacetylation) in H3K9me3 and H3K9me2 and PD is associated with paraquat (differential acetylation), manganese (hypoacetylation), and dieldrin (hyperacetylation). Discussion. Known epigenetic biomarkers associated with NDs may be identifiable in cells or cell-free DNA shed into liquid biopsy and wastewater. Improved geospatial tagging of epigenetic biomarkers and factor exposure studies will lead to earlier interventions through epigenetic biomarker detection using WBE methodology.
Sunday
3768403 - Aspartic Acid Residues are Potential Mediators of Extension in Type 51 Refractile Inclusion Bodies | Poster Board #3007
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Type 51 refractile bodies (R bodies) are enormous protein constructs made by Caedibacter taeniospiralis bacteria that rapidly and reversibly extend in response to changes in pH. Preliminary comparative analysis of carbon-carbon correlation MAS NMR spectra collected for the contracted and extended conformations identifies aspartic acid residues as potential mediators of extension. This hypothesis is supported by new data that measured the conversion pH of Type 51 R bodies to be 5.36 ± 0.06, which is very near the pKa of aspartic acid compared to other residues, and also by the high number and relatively even distribution of these residues within monomers. Studies on the mechanism of R body extension are important as they are studied in the contexts of bioengineering and medicine.
Sunday
3768407 - Stress granule marker protein TIA-1 binds preferentially to sequences optimized for both RRM2 and RRM3 | Poster Board #3009
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
TIA-1 is an RNA-binding protein that is involved in eukaryotic stress response. Under conditions of cellular stress, TIA-1 induces the formation of stress granules by binding to mRNA-preinitiation complexes. Structurally, TIA-1 has three RNA-binding domains – RRM1, RRM2, and RRM3 – as well as an intrinsically disordered domain. Previously, we found that a 30 nucleotide (nt) DNA sequence with three binding sites induced more significant aggregation when compared to a corresponding 10 nt DNA sequence with a singular binding site, and that the protein to DNA binding ratio is approximately 3:1. In this study, we tested aggregation levels, binding affinity, and protein to DNA ratios for various DNA sequences. We found that out of all tested sequences, those with adenine linkers between binding sites induced more aggregation, as well as those with T-rich and C-rich sequences optimized for RRM2 and RRM3, respectively. Additionally, we found that both the shorter linker sequences and the sequences optimized for both RRM2 and RRM3 had a higher binding affinity for TIA-1, compared to sequences that had either the longer linkers or no linkers at all.
Sunday
3768458 - The role of eIF4A in mRNA recruitment | Poster Board #3011
07:00pm - 09:00pm USA / Canada - Central - August 21, 2022 | Location: Hall F2 (McCormick Place Convention Center)
Division: [BIOL] Division of Biological Chemistry
Session Type: Poster - In-person
Accurate and efficient translation of mRNAs into proteins is essential for the existence of life. Consequently, dysregulation of translation is causally implicated in many human diseases, most prominently cancer, and is a hallmark of viral infections. The efficient and accurate selection of an mRNA and its loading onto the ribosome are regulated by a set of proteins and protein complexes known as eukaryotic translation initiation factors (eIFs). One of these factors, eIF4A, is crucial in preparing the mRNA for binding to the ribosome. While the general role of eIF4A as a helicase enzyme has previously been well studied, its precise interactions with other eIFs, the ribosome, and the mRNA during translation initiation remain poorly understood. Using ensemble fluorescence anisotropy and single-molecule fluorescence resonance energy transfer (smFRET) experiments, I aim to determine the precise interactions of eIF4A with other eIFs, the ribosome, and the mRNA. To facilitate these experiments, I have designed, constructed, over-expressed, and purified two ‘cysteine-lite’ eIF4A variants on which I have engineered a different surface-accessible cysteine onto each. These variants have enabled me to site-specifically label eIF4A with a cysteine-reactive fluorophore. With a site-specifically, fluorophore-labeled eIF4A in hand, I will initially use fluorescence anisotropy experiments to determine the equilibrium dissociation constants (Kds) governing the interactions of eIF4A with other eIFs and the mRNA, and use ATPase assays to determine which eIFs enhance the helicase activity of eIF4A. Understanding how other eIFs modulate the stability of eIF4A binding to the mRNA and, ultimately, the kinetics with which eIF4A unwinds the mRNA will allow us to better understand the role of eIF4A in mRNA recruitment and translation initiation. The results of these studies promise to provide new targets and strategies for treating diseases that arise from the dysregulation of translation initiation.