| dc.description.abstract | Lysophospholipase enzymes, lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2), are serine hydrolases responsible for the metabolism of three separate classes of bioactive lipid substrates, including lysophospholipids, prostaglandin glyceryl esters (PGG), and the S-palmitoylcysteine post-translational modification of proteins. This diverse range of substrates suggests a regulatory role of LYPLAs in a number of associated physiological processes. To understand the structural determinants of LYPLA substrate selectivity, we employed x-ray crystallography resulting in the first crystal structure of LYPLA2. These structural investigations reveal key structural differences between the enzymes, specifically in potential substrate-binding regions and sites of post-translational modification. Site-directed mutagenesis was employed to investigate these structural features with recombinant protein. Furthermore, Lypla1-/-, Lypla2-/- and double knockout neuroblastoma cell models were developed using CRISPR-Cas9 technology to investigate the activity of LYPLAs toward each type of substrate in a cellular setting. Extensive lipidomics was performed to identify changes in lipid metabolism and signaling, and LYPLA-dependent phenotypic changes were characterized in the knockout cells. Acyl-protein thioesterase activity in these cells was also assessed using alkynyl-palmitic acid and click chemistry to label palmitoylated proteins. These data suggest modest increases in global protein palmitoylation in double knockout cells and preliminary experiments have identified multiple lipoprotein substrates of LYPLAs. Here we describe the first kinetic and cellular analyses of LYPLAs’ ability to hydrolyze a range of PGG, lysophospholipid, and lipoprotein substrates. Collectively, these data provide the first structural interpretation of the molecular interactions of LYPLAs with their lipid substrates. | |
