BIOCHEMICAL CHARACTERISATION OF ABHD14A: AN ORPHAN PROTEIN

Abstract

The advent of genome sequencing technologies has revealed the presence of a myriad of proteins in the mammalian system, many of which are poorly characterised. The presence of these orphan proteins imply that our understanding of cell and tissue metabolism and its associated biochemical pathways is still dismal, underscoring the roles of these additional molecular players. Therefore, the functional annotation of these orphan proteins provides great avenues to understand their biochemistry and their physiological implications in health and disease. Enzymes of the serine hydrolase family are of particular interest in this regard, since members of this family metabolize a wide range of physiological substrates including peptides and proteins, lipids and small molecules, with a large fraction of these still unannotated. Alpha beta hydrolase domain containing protein 14A is one such uncharacterised enzyme. In our study, we sought characterisation with first identifying sequence determinants for the efficient annotation of the protein across organismic classes. We have established protocols for purifying this scarcely researched protein and used chemo proteomic methods to assay its biochemical activity, with emphasis on similar activity on induced expression in cultured cells. We also predict hydrolase activity using preferred substrates. To further investigate the roles of ABHD14A, we developed an antibody against the purified protein and characterised it using different experiments. Overall, using the novel standardised methods for assessing the activity of ABHD14A, we have tried to understand its role in mammalian biochemistry, by probing diverse aspects, including its subcellular localisation, spatiotemporal expression and metabolic changes upon induced expression.