PNPLA-mediated lipid hydrolysis and transacylation - At the intersection of catabolism and anabolism.

Patatin-like phospholipase domain containing proteins (PNPLAs) play diverse roles in lipid metabolism. In this review, we focus on the enzymatic properties and predicted 3D structures of PNPLA1-5. PNPLA2-4 exert both catabolic and anabolic functions. Whereas PNPLA1 is predominantly expressed in the epidermis and involved in sphingolipid biosynthesis, PNPLA2 and 4 are ubiquitously expressed and exhibit several enzymatic activities, including hydrolysis and transacylation of various (glycero-)lipid species. This review summarizes known biological roles for PNPLA-mediated hydrolysis and transacylation reactions and highlights open questions concerning their physiological function.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.