Cellular and molecular mechanisms involved in the biosynthesis and hydrolysis of the endocannabinoid 2-arachidonoyl-glycerol and its congeners

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Arachidonic acid (AA) is fatty acid mainly found in animals. It plays critical roles in many processes, from fever to reproduction. AA is so important that animals unable to synthesize it, such as the cat, are obligatory carnivorous to obtain the AA from the meat they eat. Humans can however make it from vegetable oils containing linoleic acid. In our body, most of the AA is stored within our cell membranes. Multiple enzymes are responsible for storing AA within our cell membranes and for providing free AA on demand. AA is the precursor of numerous eicosanoids such as prostaglandins and leukotrienes, which participate to a myriad of physiological processes. AA is also the precursor of the main endocannabinoids, which mimic some of the physiological effects of cannabis, as well as displaying many other regulatory functions throughout our organism. Thus, it is not surprizing that AA levels are tightly controlled. Furthermore, many enzymes are involved in the degradation of AA-containing endocannabinoids, although the involved proteome is not completely defined. My NSERC research program focuses at understanding how arachidonic acid (AA) metabolism occurs in inflammatory cells and to define the regulatory mechanisms involved in AA turnover and release. This is important given the important role AA has at regulating the abovementioned functions, notably during the course of inflammation. We recently showed that the fate of AA is diverse in human neutrophils: 1) it is partly transformed into eicosanoids; 2) it is rapidly incorporated into phospholipids. Of note, 3) some of the reacylated AA is rereleased as 2-arachidonoylglycerol (2-AG), an endocannabinoid; While the first two events had previously been documented us and others, we are the first to document the release of 2-AG. The AA-induced 2-AG biosynthesis is prevented by Acyl-CoA synthetase and Acyl-CoA tranferase inhibition, indicating the remodeling of AA into phospholipids. To be adequately observed, we must eliminate the ability of leukocyte to hydrolyze 2-AG. For the next five years, we propose to define, at the cellular and molecular level, wether additional substrates participate to the biosynthetic pathway we unmaked. We will thus investigate numerous unsaturated fatty acids and oxylipins. Finally, and as underscored above, 2-AG is very labile and is hydrolyzed very rapidly (seconds to minutes). While defining which 2-AG lipases were expressed by neutrophils, we pinpointed a new 52 kDa protein not documented for hydrolyzing 2-AG. We will thus identify that protein by mass spectrometry. Once identified, we will confirm using recombinant proteins the selectivity and specificity of the enzyme. In conclusion, this project will provide important insights on the regulation of AA and AA-derived effectors. It will also provide useful data for the understanding of lipid mediator biosynthesis and their role in physiology and will confirm AA as a central regulator of 2-AG synthesis.