Lipases in the Endocannabinoid System

Lipases in the Endocannabinoid System The endocannabinoid system (ECS) is found in the central nervous system (CNS) and includes the cannabinoid receptors CB1 and CB2 [the ‘locks’], the endogenous ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG)[the ‘keys’], and the enzymes (‘locksmiths’) that make and break down these ‘keys’. Activation of the CB1 and CB2 receptors by the endocannabinoid ligands 2-AG and AEA regulates neurotransmission and neuroendocrine activity in the brain. In the brain, 2-AG is found at levels approximately 170 times higher than that of AEA making it the most abundant endocannabinoid ligand. In mouse models for Parkinson’s disease (PD), it was observed that an increase of 2-AG levels suppressed inflammatory cytokines. The relationship of 2-AG levels to the enzymes that synthesize and degrade it make 2-AG and its enzymes excellent targets for biotherapeutics. In this study, we examine the role of the principal synthetic enzyme, diacylglycerol lipase-ɑ (DAGL-ɑ) and the principal hydrolytic enzyme monoacylglycerol lipase (MAGL). Through studies of these lipases, we hope to provide further insight into 2-AG signaling in disease states. There were two goals of this thesis. The first goal was to elucidate DAGL-ɑ by expressing, purifying, and characterizing its enzymatic activity. We used an SDS-PAGE gel which separates proteins by size and found two bands which we believe represent DAGL-ɑ at~120 and 80 kDa in size. In the future we hope to optimize the purification method so that we have enough DAGL-a to perform structural studies. The second goal was to investigate the mechanisms of activity of MAGL. MAGL is a well characterized enzyme whose structure was solved in 2010. Given that MAGL is responsible for regulating the levels of 2-AG, there is interest in designing inhibitors to MAGL to treat neurodegenerative diseases. In this experiment, we looked at how different changes in the structure will affect the enzyme activity. By identifying interesting changes in activity, we hope to infer on the structural mechanism of MAGL activity. Our results demonstrated a mechanism involving activated states which changes the enzymatic activity of MAGL. By examining these mechanisms, we hope to identify potential drug targets involving MAGL. In summary we believe we have contributed to the understanding of 2-AG regulation by DAGL-ɑ and MAGL which is useful in designing therapies for neurodegenerative diseases. Master’s Degree Project in Molecular Biology 60 credits 2020 Department of Biology, Lund University Advisor: Christian Krintel Lund University/Department of Structural Biology (Less)