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

内源性大麻素 2-花生四烯酰甘油及其同系物生物合成和水解涉及的细胞和分子机制

• 批准号: RGPIN-2021-03777
• 负责人: Flamand, Nicolas
• 金额: $ 3.06万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760659
• 关键词: Cellular; molecular; mechanisms; involved; biosynthesis

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.

花生四烯酸（AA）是一种主要存在于动物体内的脂肪酸。它在许多过程中起着关键作用，从发烧到繁殖。AA是如此重要，以至于无法合成它的动物，如猫，是强制性的食肉动物，从他们吃的肉中获得AA。然而，人类可以从含有亚油酸的植物油中制造它。在我们的身体中，大部分AA储存在我们的细胞膜内。多种酶负责在我们的细胞膜内储存AA，并根据需要提供游离AA。AA是许多类二十烷酸的前体，如白藜芦醇和白三烯，其参与无数的生理过程。AA也是主要内源性大麻素的前体，它模仿大麻的一些生理作用，并在整个生物体中显示许多其他调节功能。因此，严格控制AA水平并不奇怪。此外，许多酶参与含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研究计划的重点是了解花生四烯酸（AA）代谢如何在炎症细胞中发生，并确定参与AA周转和释放的调节机制。这是重要的，因为AA在调节上述功能方面具有重要作用，特别是在炎症过程中。我们最近发现AA在人类中性粒细胞中的命运是多样的：1）它部分转化为类花生酸; 2）它迅速掺入磷脂。值得注意的是，3）一些再酰化的AA被重新释放为2-花生四烯酸甘油（2-AG），一种内源性大麻素;虽然前两个事件之前已经被我们和其他人记录，但我们是第一个记录2-AG释放的人。AA诱导的2-AG生物合成被酰基辅酶A合成酶和酰基辅酶A转移酶抑制剂阻止，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，我们打算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。最后，如上所述，2-AG非常不稳定，水解非常迅速（数秒至数分钟）。在确定哪些2-AG脂肪酶由中性粒细胞表达的同时，我们确定了一种新的52 kDa蛋白质，该蛋白质未被记录用于水解2-AG。因此，我们将通过质谱法鉴定该蛋白质。一旦鉴定，我们将使用重组蛋白确认酶的选择性和特异性。总之，该项目将提供重要的见解AA和AA衍生的效应器的调节。它也将提供有用的数据，了解脂质介质的生物合成及其在生理学中的作用，并将确认AA作为2-AG合成的中央调节器。