Introduction: Histidine triad nucleotide binding (HINT) proteins are the most ancient members of the histidine triad (HIT) protein, the superfamily of nucleotidyltransferases and hydrolyases, which are highly conserved and widely distributed in eukaryotic and prokaryotes. Defined by the highly conserved HXHXHXX functional domain (where X denotes a hydrophobic amino acid), these proteins, particularly human HINT1, play crucial roles in human pathology. Functioning as a tumor suppressor, HINT1 is linked to neuropsychiatric disorders like Intellectual and Developmental Disabilities (IPNs). Despite these insights, the endogenous substrate(s) of HINT1 remain elusive. HINT exhibits potent catalysis, hydrolyzing acyl-AMP to generate a carboxylic acid and AMP in vitro, with these intermediates containing amino acids such as lysine, methionine. Through detailed in vivo and in vitro studies of HINT function, it has been suggested that HINT may regulate translation fidelity as a pre-transfer editing enzyme, thus serving as a supplementary mechanism to ensure the accuracy of the translation editing process.

Preliminary data: Upon conducting the knock-out procedure, it was observed that bacterial knock-out strains exhibited a decelerated growth rate, a phenotype intricately linked to modifications in the active site. In the case of C. elegans, knock-out worms displayed a diminished brood size, which we further attributed to a decrease in embryo hatching rates, ceasing after the C. elegans embryos reached the 3-fold stage. Subsequently, employing functional proteomics, we identified an elevated protein translational error rate following hinT gene knock-out in both E. coli and C. elegans, in comparison to the effect induced by a drug known to cause translational errors. Further scrutiny led to the identification of mis-translated proteins, which will undergo validation using an E. coli cell-free system. Through protein-protein interaction experiments, specific aminoacyl-tRNA synthetases, have been characterized. Collectively, these experiments underscore the widespread distribution and ancient nature of HinT, which modulates physiological functions by controlling translation fidelity as a pre-transfer editing enzyme. This advancement holds promise for enhancing research into HinT-related pathologies, including cancer and neuropsychiatric disorders.

Tsui-Fen Chou

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