HUMAN ABHD14B: A NOVEL LYSINE DEACETYLASE REGULATING TRANSCRIPTION OF METABOLIC GENES

Abstract

My research involves the biochemical characterization of ABHD14B and finding its biological substrate and function. ABHD14B is a metabolic serine hydrolase that was discovered as an interactor of the HAT domain of the TAFII250 protein, the largest subunit of general transcription factor TFIID. The crystal structure of ABHD14B was resolved and found to contain the canonical alpha/beta-hydrolase domain and the conserved catalytic triad of the serine hydrolase superfamily. The enzyme was shown to be hydrolytically active and possess a possible role in transcriptional activation. ABHD14B has been implicated in various diseases including cancer, making it important to study this enzyme. Following up on this study I first purified the recombinant human WT and active site S111A mutant ABHD14B and showed that the mutant is catalytically inactive. I also showed that ABHD14B prefers shorter substrates for hydrolysis such as pNP-acetate as compared to pNP with long-chain acylations and that it transfers the hydrolyzed acetate group onto a CoA molecule to form acetyl-CoA. I successfully generated stable genetic knockdowns of ABHD14B in the HEK293T cell line. In these knockdown lines, I showed an increase in the protein lysine acetylation and a concomitant decrease in the acetyl-CoA levels. This led me to hypothesize acetylated lysine residues of proteins to be the biological substrates of ABHD14B. I successfully validated this hypothesis using acetylated peptides and proteins as hydrolysis substrates and showing the acetyl-CoA formation. Therefore, ABHD14B deacetylates post-translationally acetylated lysine residues and transfers the acetate group onto a CoA molecule to form acetyl-CoA. Hence, I annotated ABHD14B as a novel lysine deacetylase as its mechanism is distinct from the other two known lysine deacetylases, sirtuins, and HDACs. Next, I showed that depletion of ABHD14B leads to significant changes in transcriptional regulation of metabolic genes especially those involved in glucose metabolism (glycolysis, TCA cycle, ETC, gluconeogenesis, etc.,). In correlation to this, I showed significant dysregulation in the cellular levels of intermediates of glycolysis and TCA cycle in ABHD14B knockdown HEK293T cells. Overall, my research describes for the first time that ABHD14B is a novel lysine deacetylase that regulates the transcription of metabolic genes and thereby glucose metabolism. Going ahead the protein substrates of this deacetylase and its specific role in metabolism at systemic levels need to be investigated.