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

花生四烯酸（AA）是一种主要存在于动物体内的脂肪酸。它在从发热到繁殖的许多过程中起着关键作用。AA是如此重要，以至于不能合成它的动物，如猫，是强制性的食肉动物，以从它们所吃的肉中获取AA。然而，人类可以从含有亚油酸的植物油中获得亚油酸。在我们体内，大部分AA储存在细胞膜内。多种酶负责将AA储存在细胞膜内，并根据需要提供免费的AA。AA是许多类二十烷的前体，如前列腺素和白三烯，它们参与了无数的生理过程。AA也是主要内源性大麻素的前体，内源性大麻素模仿大麻的一些生理作用，以及在我们的生物体中显示许多其他调节功能。因此，AA等级受到严格控制也就不足为奇了。此外，许多酶参与了含有aa的内源性大麻素的降解，尽管所涉及的蛋白质组尚未完全确定。我的NSERC研究项目侧重于了解花生四烯酸（AA）代谢如何在炎症细胞中发生，并定义参与AA周转和释放的调节机制。鉴于AA在调节上述功能方面的重要作用，特别是在炎症过程中，这一点很重要。我们最近发现，AA在人类中性粒细胞中的命运是多种多样的：1)它部分转化为类二十烷酸；2)它被迅速并入磷脂。值得注意的是，3)一些再乙酰化的AA被释放为2-花生四烯醇甘油（2-AG），一种内源性大麻素；虽然我们和其他人之前已经记录了前两个事件，但我们是第一个记录2-AG发布的人。抑制酰基辅酶a合成酶和酰基辅酶a转移酶抑制了AA诱导的2-AG生物合成，表明AA重塑为磷脂。为了充分观察，我们必须消除白细胞水解2-AG的能力。在接下来的五年里，我们将在细胞和分子水平上确定是否有其他底物参与我们所发现的生物合成途径。因此，我们将研究许多不饱和脂肪酸和氧化脂类。最后，如上所述，2-AG非常不稳定，水解速度非常快（几秒到几分钟）。在确定哪些2-AG脂肪酶是由中性粒细胞表达的同时，我们确定了一种新的52 kDa蛋白，该蛋白未被记录用于水解2-AG。因此，我们将通过质谱法鉴定该蛋白质。一旦确定，我们将使用重组蛋白确认酶的选择性和特异性。总之，该项目将为AA及其衍生效应物的调控提供重要的见解。这也将为了解脂质介质的生物合成及其在生理中的作用提供有用的数据，并将证实AA是2-AG合成的中心调节剂。

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.