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Emerging Areas and New Methods in Biological Chemistry:
08:00am - 12:00pm USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Phoebe Glazer, Organizer, Presider; Ekaterina Pletneva, Organizer
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Division/Committee: [BIOL] Division of Biological Chemistry
Tuesday
3643870 - Effects of N-linked glycans on the stability of the spike protein in SARS-CoV-2: Molecular dynamics simulations
08:00am - 08:15am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Spike Protein in SARS-CoV-2 is extensively decorated with heterogeneous N-linked glycans hanging from the trimer surface that are important for folding and “for modulating accessibility to host proteases and neutralizing antibodies” and so the existence of glycans is believed to be one of the reasons why the coronavirus has caused a large number of infections and mortality. In this study, we perform all-atom molecular dynamics simulations of closed form of SARS-CoV-2 Spike (6VXX.pdb) without and with the N-linked glycans to understand the effects of these glycans on the stability of the spike glycoprotein in SARS-CoV-2. All the calculations were carried out using the GROMACS 2019 code and the GROMOS 54a7 force field extended to include the N-linked glycans as pseudo-amino acids. After a 100 ns of simulation on the spike proteins without and with the N-linked glycans, we found that the presence of glycans increases the local stability in their vicinity; even though their effect on the full structure is negligible.

Tuesday
Dirigent proteins (DPs) were first discovered over two decades ago from the plant Forsythia × intermedia. Later, other DPs were discovered, and sequence analysis suggested that DP homologs have several distinct sub-families. All known DPs mediate stereo-selective coupling of plant phenols. Recent studies have demonstrated the potential of DPs for highly efficient in vitro stereo-selective synthesis of natural product analogs not easily achievable with conventional chemical approaches. In vascular plants, DPs hypothetically function, along with other essential enzymes/proteins (e.g. oxidases), as part of lignin/lignan forming complexes (LFCs). However, very limited experimental data are available to elucidate the putative LFC. Part of the challenges is the lack of sequenced genome of the Forsythia plant where the original DP was purified from. Herein, we used de novo sequencing mass spectrometry to identify other proteins in a DP-enriched solubilized protein fraction, via adaptation of our initial report of DP solubilization and purification.

With the help of transcriptome and genome data from homologous plants, we confidently identified 14 proteins in the fraction, including two new DP homologs not previously reported. Native mass spectrometry of the same fraction showed they also formed hetero-trimers. Molecular dynamics simulations suggest that similar hetero-trimers were possible between DP homologs with comparable sequence similarity. Other identified proteins in the DP-enriched preparation were putatively associated with DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for their associations in vivo, none were found to form long-lived complexes with DPs under the specific experimental conditions we have explored. We are currently developing new purification workflows to better capture the putative complexes. At the same time, we are expanding our study to other DP homologs in model plant organisms using our integrated mass spectrometry method. We believe our study will reveal novel molecular mechanisms of DP for understanding of biosynthesis of lignans and lignins.

Tuesday
3649910 - Chemical tools for studying non-conventional protein phosphorylations
08:30am - 08:45am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Despite the well-recognized biological importance of protein phosphorylation, non-conventional phosphorylations on histidine and arginine residues have evaded our attention and scrutiny for a long time. This lack of knowledge stems from the inherent chemical instability of phosphohistidine (pHis) and phosphoarginine (pArg), which makes the analysis of these phosphorylations notoriously challenging.
Here we present our recent progress in developing fluorescent sensors to monitor the activity of the kinase and phosphatase that regulate these elusive forms of phosphorylation. Both synthetic and genetically encoded sensors have been successfully utilized for convenient real-time measurement of the target enzyme activities.

Tuesday
3654393 - Fatty acid synthases (FASs) enable access to new-to-nature compounds
08:45am - 09:00am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Martin Grininger, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Multienzyme type I fatty acid (FASs) and polyketide synthases (PKSs) catalyze C-C bond forming reaction in compartmentalized space. Compartmentalization is largely achieved by the transacylation function of FASs and PKSs, i.e. the enzymatic reaction responsible for selecting substrates from the bulk cytoplasm, and further relies on a specific protein architecture as well as the shuttled distribution of substrates between the enzymatic domains. Controlling the transacylation function of FASs and PKSs is a powerful mean to modulate the product output of these proteins for access to technologically and medicinally relevant compounds.
Two examples for transacylation-based engineering will be presented in this talk: (i) We employed fungal FAS for the custom synthesis of commodity products, among them short-chain fatty acids, methylketones and lactones. For increasing complexity of synthesized compounds, FAS constructs were modulated in transferase function for acting in sequence. (ii) The mammalian FASs features high transacylation rates for a wide range of substrates. We inserted the mammalian FAS transferase domain (MAT) into modular PKSs to enable the in vitro semisynthesis of new polyketides, including fluorinated polyketides with a macrolide scaffold similar to the next-generation antibiotic solithromycin (2-Fluoro-YC-17, see Figure).
Semisynthetic approach to first produce the fluoro-methyl disubstituted macrolactone, which is then biotransformed to the fluorinated derivative of the antibiotic YC-17 using strain DHS316.

Semisynthetic approach to first produce the fluoro-methyl disubstituted macrolactone, which is then biotransformed to the fluorinated derivative of the antibiotic YC-17 using strain DHS316.


Tuesday
3651098 - Histone modifications in ALS/FTD: New avenues for suppressing toxicity of neurodegenerative disease proteins
09:00am - 09:15am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease that affects cells in the brain and the spinal cord. frontotemporal dementia (FTD) is another disorder involving progressive neuronal loss in the frontal and temporal lobes of the brain. ALS and FTD form a neurodegenerative continuum and share pathological and genetic features. ALS/FTD has been linked to mutations in many genes including FUS, TDP- 43 and C9orf72. Interestingly, the protein products of these genes accumulate in inclusions within affected neurons.
Eukaryotic DNA is packaged into chromatin, a highly organized protein-DNA complex. Changes in the structure of chromatin are sufficient to cause heritable phenotypic changes termed epigenetic. Epigenetic mechanisms include the methylation of DNA and the covalent post-translational modification of histone proteins.
We have discovered distinct histone modification profiles associated with FUS, TDP-43 and C9orf72 proteinopathies in yeast and human cell models. Furthermore, we demonstrate that interfering with histone modification changes can counter the repercussions of protein aggregation on cell survival. Our data raises the novel hypothesis that the toxic effect of protein aggregation in neurodegeneration is related to its association with altered histone marks. Altogether, our findings highlight novel epigenetic mechanisms at play in ALS/FTD. Epigenetic processes are highly accessible targets for pharmaceutical treatments and thus they can lead to novel, alternative approaches in the treatment of ALS/FTD and other neurodegenerative diseases.

Tuesday
3649542 - Rationally designed molecular prosthetic for cystic fibrosis
09:15am - 09:30am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
We have recently shown that ion channels formed by the natural product amphotericin B can operate like prostheses on the molecular scale to replace missing CFTR anion channels in the noses of people with cystic fibrosis (CF). Here we report an extensive series of structural studies, including high-resolution magic-angle spinning solid-state NMR experiments in concert with simulated annealing and molecular dynamics computations, as well as mechanistic, biophysical, and nanoparticle engineering studies that have collectively enabled the rational design of a dry powder inhaler for delivering this molecular prosthetics approach to the airways of people with CF. For example, we illuminated key structural features of extramembranous sponges formed by AmB that help explain how this biologically active clathrate hosts sterol guests and suggest an equilibrium with small intramembranous ion-channel aggregates. These structural studies in concert with a range of complementary biophysical and mechanistic studies enabled the rational development of a novel dry powder inhaler that leverages fundamental insights into the role of cholesterol to effectively separate the ion channel activity of AmB from its undesired cytocidal sponge-like action. A suite of preclinical studies suggests that this novel inhaled formulation may serve as an alternative treatment for people with CF, including for those who cannot benefit from CFTR modulators
Tuesday
3651749 - Developing targeted covalent inhibitors of lipid enyzmes through discovery of cryptic binding sites
09:30am - 09:45am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Our group developed sulfonyl-triazoles as a new reactive group for covalent binding to catalytic and non-catalytic tyrosine sites on proteins through sulfur-triazole exchange chemistry (SuTEx). Here, we describe the application of SuTEx for fragment-based ligand discovery in live cells to develop a prostaglandin reductase 2 (PTGR2) ligand. PTGR2 reduces 15-keto-PGE2 into 13,14-dihydro-15-keto-PGE2 lipid, which are critical for inflammatory and cancer signaling. We show by quantitative chemical proteomics that SuTEx fragment compounds ligand several tyrosine sites in the active site of PTGR2. Medicinal chemistry optimization produced first generation PTGR2 covalent inhibitors that show promising activity in live cells. While PTGR2 ligands are directed towards tyrosines located in the active site, we show in complementary studies that sulfonyl-triazoles are capable of binding regions remote from catalytic domains of lipid kinases by inclusion of appropriate recognition elements. We conclude with a discussion of how these cryptic lipid binding sites can be leveraged for development of selective inhibitors against related members of lipid enzyme superfamilies.
Tuesday
Intermission
09:45am - 10:00am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid

Tuesday
3659882 - Insights into quantitative proteomics
10:00am - 10:15am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Rapidly evolving mass spectrometry (MS)-based technologies in proteomics enable to study proteins in a wide range of samples starting from a single cell to a complex biological sample such as blood and tissue. Targeted proteomics is a powerful tool for hypothesis testing for a wide range of diseases including cancer, inflammatory diseases, cardiovascular diseases, diabetes and even psychiatric disorders.. The peptide-centric technique focuses on identifying and/or quantifying precisely the protein's unique peptide(s) using MS in bottom-up proteomics. Here, we investigated the protein-peptide relationship from quantitative analysis perspective. Alpha-2-macroglobulin (A2MG), clinically important protein linked to liver, lung, neurological, and prostate cancer, was selected as a reference protein. Unique peptides representing A2MG were monitored using triple quadrupole (QQQ) MS operated in multiple reaction monitoring (MRM) mode. Protein-peptide and protein-glycopeptide correlations, digestion efficiency, matrix, and concentration-effect were examined using statistical approaches. The preliminary results suggest that the selection of the unique peptide is the most critical step in targeted proteomics as each peptide behaves differently depending on the digestion conditions. The outcomes of this research will provide insights into quantitative proteomics.
Tuesday
3656147 - Single mRNAs and protein biomolecules counting using solid-state nanopores, from basic science to clinical applications
10:15am - 10:30am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Amit Meller, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
SARS-CoV-2 outbreak of the coronavirus disease (COVID-19) has underlined the acute need for extremely sensitive, accurate, fast, point-of-care mRNA quantification sensors. Here I will show how solid-state nanopores can be used to digitally count target mRNA molecules from both biological and clinical Covid-19 samples surpassing the accuracy and gold-standard” RT-qPCR. Additionally, we applied our method for the sensing of cancer metastatic mRNA biomarkers MACC1 and S100A4 at early stage of the diseases, suggesting a potential use of the method in early precision medicine diagnostics.
Moving beyond nucleic acids, I will discuss our group's progress towards whole proteome sensing using nanopores and nanochannels. Chemical labelling of two or three amino acids in full-length proteins, combined with single-molecule fluorescent sensing allows "single shot" identification and protein counting of selected target proteins in biological samples, as the they translocate through a solid-state nanopore device or migrate in a custom nano-channel. Our research opens up vast directions for clinical protein identification, as well as for single-cell proteomics of even rarely expressed proteins.

Tuesday
3651018 - General design of caging-group free photoactivatable fluorophores for live-cell nanoscopy
10:30am - 10:45am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Superresolution microscopy (fluorescence nanoscopy) techniques have revolutionized our ability to visualize nanoscale biological systems in a minimally-invasive manner. The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution technique, and the discovery of new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable or caged dyes (in which the off–on transition is irreversible and triggered by light) offer significant improvements to many superresolution techniques, but often make use of photolabile protecting groups, resulting in their poor solubility and membrane impermeability. Caging-group free, compact photoactivatable and biocompatible fluorophores are thus highly desirable in fluorescence nanoscopy applications, enabling lower molecular weight labels, provided that the photoactivation is rapid, complete and byproduct-free.

Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These compounds assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs yielding a family of photoactivatable labels that span much of the visible spectrum. This new family of dyes displays excellent live-cell compatibility, a resilience to cellular nucleophiles, and a noteworthy photostability. We evaluated the utility of these photoactivatable dyes and labels in both fixed and live cell labelling strategies, and further demonstrated their performance in optical microscopy and nanoscopy techniques including STED, PALM, and MINFLUX.

Tuesday
Methyltransferases (MTases) catalyze targeted methyl group transfers from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) to a multitude of biological targets in the cell. Methylation of cytosine and adenine residues in DNA is part of epigenetic regulation of gene function in vertebrates and can often be associated with the occurrence and progression of human disease. We went on to redesign the methyltransferase reactions for targeted transfer of larger chemical moieties by employing synthetic AdoMet analogues in which the sulfonium-bound methyl group is replaced by an extended side chain that typically carries a transferable propargylic moiety, a linear linker and a terminal reporter/functional group. Using a series of such AdoMet analogues and natural or engineered variants of appropriate MTases as targeting vehicles we achieved precise covalent functionalization and labeling of DNA. Practical utility of this labeling platform was demonstrated by single molecule sub-megabase scale genotyping and economical DNA epigenome profiling in vitro and ex vivo. Furthermore, we CRISPR-engineered the Dnmt1 locus in mouse embryonic stem cells and embryonic fibroblasts to install the engineered codons, which, following pulse-internalization of the synthetic cofactor analog AdoX by electroporation, permitted selective azide-tagging of Dnmt1-specific genomic targets in vivo. The deposited chemical groups can be exploited as click handles for precise mapping of the tagged methylation sites in the genomic sequence or spatial visualization in fixed nuclei, enabling selective high-resolution temporal genome-wide tracking of the Dnmt1 catalysis in live mammalian cells (Fig.1). Altogether, these engineered chemically-controllable systems offer unprecedented inroads into deciphering and altering the mammalian epigenetic mechanisms and facilitate the development of next generation diagnostics.
Figure 1.

Figure 1.


Tuesday
3659240 - QM-cluster model study of enzymatic reaction between cytochrome P450 enzyme GcoA and various substrates OnDemand
11:00am - 11:15am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Recently, a promising Amycolatopsis cytochrome P450-reductase pair -- a two component P450 class composed of GcoA and GcoB has been discovered.1 GcoA was found to efficiently demethylate several lignin-derived monomers,2suggesting a broad substrate scope for lignin degradation and valorization. The key step of the O-demethylation of guaiacol by GcoA was studied by our group with Density Functional Theory using various sized quantum mechanical (QM)-cluster models.3 The computed free energy of activation is in good agreement with experimentally measured kcat of demethylation of guaiacol by GcoA (6.8 ± 0.5 s-1 at 25°C). Hence, the enzymatic reactions of seven different substrates catalyzed by GcoA were studied by a similar approach, starting from Molecular Dynamics simulations of GcoA in complex with each substrate. QM-cluster models were generated by the toolkit Residue Interaction Network-based ResidUe Selector (RINRUS). This study will provide details for better understanding enzymatic O-demethylation of lignins to form catechol derivatives by GcoA and shine a light on structure-activity relationship models for future ligand design and enzyme bioengineering.
Tuesday
3662153 - Employing chemical genetics to develop Insect glia as a cellular target for insecticides
11:15am - 11:30am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Daniel Swale, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
The development of novel insecticide targets has been of consistent interest to the field of insecticide science, yet few new biochemical targets have emerged over the past two decades. Insect neurons are the target tissue for >85% of commercialized insecticides, thus it is surprising the functional roles of insect glial cells remain poorly understood and there are no glia-directed insecticides. Recent work has shown expression of various ion channels in insect glia cells, raising the intriguing possibility that glial cells contribute to neuronal function of insects and may represent a cellular target for insecticide design. Considering this, we tested the hypothesis that inwardly rectifying potassium (Kir) channels expressed in glia cells contribute to nerve firing and Kir inhibition will have deleterious consequences to neuronal function. To test this hypothesis, we used CRISPR/Cas9 in Drosophila to fluorescently tag the endogenous gene loci of Kir1, Kir2 and Kir3. Fluorescent microscopy imaging showed Kir2 channel subunits are localized at the membrane of glial cells, but not neurons. Functional studies support this localization pattern as patch-clamp electrophysiology showed an inward K+ conductance of 104 ± 60 pA/pF in Drosophila glia cells but no significant inward K+ current in central neurons. These data led us to speculate Kir channels constitute a mechanism for rapid clearance of K+ ions from the extracellular space during neuronal activity and thus, inhibition of glial Kir channels would result in membrane depolarization and increased firing. To test this, we performed extracellular recordings of Drosophila descending neurons and found pharmacological inhibition of Kir channels significantly (P<0.05) increased the firing rate and lead to nerve death (IC50: 23 µM). Importantly, inhibition of glial cell function with Kir specific pharmacophores resulted in acute lethality that highlights the potential for developing novel mode- and mechanism- insecticides targeting glial cell function.
Tuesday
3648860 - New opportunities for studying the function of the nucleosome remodeling factor, NURF
11:30am - 11:45am USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
William Pomerantz, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Protein members of nucleosome remodeling complexes are emerging therapeutic targets. One protein of interest, the Bromodomain PHD Finger Transcription Factor, BPTF, is an essential member of the human nucleosome remodeling factor NURF. In recent years years, BPTF has become increasingly identified as a pro-tumorigenic factor, prompting investigations into the molecular mechanisms associated with BPTF function. Despite a druggable bromodomain which engages in protein-protein interactions with acetylated histones, small molecule discovery is at an early stage. Our lab has developed novel screening approaches using protein-based 19F NMR, protein crystallography, and supporting biophysical methods to develop both the first inhibitor of the BPTF bromodomain, and now more potent and selective chemical probes. These molecules have been used in both cell-based assays as well as in vivo. They have demonstrated the importance of the bromodomain for mediating transcription as well as serving as a mechanism for reducing c-Myc occupancy on chromatin. Most recently they have showed synergistic effects with chemotherapeutic drugs in breast cancer models. These new inhibitors are envisioned to serve as useful chemical probes of BPTF function both in normal and pathophysiology. Finally, their potential as novel heterobifunctional molecules will also be discussed.
Tuesday
3656652 - In situ methods for capturing dynamic O-GlcNAc protein glycosylation events
11:45am - 12:00pm USA / Canada - Pacific - March 22, 2022 | Location: Sapphire I/J (Hilton San Diego Bayfront)
Charlie Fehl, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - Hybrid
Living cells continually adapt to their environments. Observing these cellular mechanisms in real-time and space is challenging. A key sugar nutrient-sensing mechanism in mammalian cells is the reversible protein modification O-GlcNAc. O-GlcNAcylation (O-linked N-acetylglucosamine addition to serine and threonine protein residues) is derived from intracellular glucose and therefore alters protein (and cell) behavior as a response to changes in local cellular environment.

Very few biological or chemical methods exist to study O-GlcNAcylation in living cells, limiting our understanding of dynamic O-GlcNAc protein modifications in changing physiological settings (a). Here, we present spatiotemporal tools to capture and perturb O-GlcNAc dynamics in living cells based on photochemistry (b) and spatial protein labeling (c). By harnessing these bioorthogonal chemical labeling reactions, our tools can identify O-GlcNAc changes in real-time as cells perform nutrient sensing and signaling activities. We identify key proteins that respond to cellular environment changes via spatiotemporal O-GlcNAc regulation, such mRNA splicing factors as well as the signal transduction factor NF-kB. Our real-time, intracellular tools reveal effects driven by O-GlcNAc cycling in situ and offer insight into cell regulatory events at the molecular level.