Molecular Basis for Membrane Binding and Lipolysis Activation by ABHD5

Triglycerides are the main reservoir for long-term energy storage in humans and are stored intracellularly in lipid droplets. Abnormal triglyceride metabolism is a key factor leading to obesity, diabetes, fatty liver disease and cardiovascular disease. When energy is needed, triglycerides are broken down in a metabolic pathway called lipolysis. Intracellular lipolysis is initiated by adipose triglyceride lipase (ATGL), which hydrolyzes triacylglycerol (TAG) to make diacylglycerol and a free fatty acid. Alpha-beta hydrolase domain-containing 5 (ABHD5) is a highly conserved regulator of ATGL-mediated lipolysis that increases ATGL activity and is necessary to maintain lipid homeostasis. Consistently, mutations in ABHD5 cause Chanarin-Dorfman syndrome, which leads to excessive accumulation of triglycerides. Despite over two decades of studies using biochemical approaches to mouse models, the structure of ABHD5 and molecular mechanisms of regulation remained uncharacterized. To understand the mechanism, we purified full-length human ABHD5, visualized its self-assembled, multiple oligomeric state in the absence of detergents by cryo-EM, and generated preliminary crystals of detergent-solubilized, monomeric ABHD5. We found ABHD5 is phosphorylated in vitro by PKA and recruited to membranes by specific lipids. We also purified the full-length human ATGL. To further investigate the structural and regulation mechanisms of ABHD5 and ATGL, we will utilize hydrogen deuterium exchange mass spectrometry to map ATGL and ABHD5 protein-protein and protein-membrane interactions, biochemical experiments to study their co-activation mechanism and membrane interaction. This study will provide a deeper understanding for the clinically relevant mutations, membrane recruitment and lipolysis activation of these key players in lipid metabolism.