Structural Biology of Oxylipin Biosynthesis

描述（由申请人提供）：目的：本提案的更广泛目标是了解不同的整合膜酶如何利用立体选择性氧化作用从一组定义的多不饱和脂肪酸（PUFA）底物中产生独特的氧脂。氧脂素是由18-22碳PUFA通过细胞色素P450、脂氧合酶和环氧合酶（考克斯-1和考克斯-2）的催化活性添加分子氧而生物合成的生物活性脂质介质。生物学上最重要的氧脂类之一是类二十烷酸类，其包括花生四烯酸衍生的白细胞三烯（leukotrienes）和花生四烯酸（arachidonic acid）。这些产物负责调节基本生理过程，并作为炎症过程和其他免疫反应的有效脂质介质。考克斯-1和考克斯-2是阿司匹林和其他非甾体抗炎药（NSAID）的药理学靶点，包括考克斯-2特异性抑制剂万络（Vioxx）、倍他乐（Bextra）和西乐葆（Celebrex）。具体目标：利用诱变、功能分析和X射线晶体学方法，我们将（1）阐明病原体诱导型加氧酶的结构，并在分子水平上表征18碳PUFA立体选择性氧化为2 R-氧脂素产物的机制和结构决定因素;（2）阐明独特的15 R-PG产生考克斯的结构：PUFA复合物，以便在分子水平上了解活性位点中PUFA的构象如何影响这些新产品生成中的立体特异性氧化。健康相关性：PUFA在健康和疾病中的作用正在引起人们的新兴趣，公众对健康食品和生活方式的看法更加集中，这些化合物在某些临床条件下具有显著影响。蛋白质，如考克斯-2，在用药理学抑制剂处理后显著改变其产物谱的能力已经导致对这些酶如何起作用的进一步研究。我们的研究将提供一个完整的机制，了解新的脂质是如何从多不饱和脂肪酸阿司匹林治疗后衍生，并提供有价值的见解，为管理关节炎和血管炎症的新的或联合治疗方法的发展，具有更少的不良副作用。

DESCRIPTION (provided by applicant): OBJECTIVE: The broader goal of this proposal is to understand how different integral membrane enzymes utilize stereoselective oxygenations to generate unique oxylipins from a defined set of polyunsaturated fatty acid (PUFA) substrates. Oxylipins are bioactive lipid mediators that are biosynthesized from 18-22 carbon PUFAs through the addition of molecular oxygen via the catalytic activity of cytochrome P450s, lipoxygenases, and cyclooxygenases (COX-1 and COX-2). One of the most biologically important groups of oxylipins is the eicosanoid class, which include prostaglandins (PGs) and leukotrienes derived from arachidonic acid. These products are responsible for the modulation of basic physiologic processes and act as potent lipid mediators of the inflammatory process and other immune responses. COX-1 and COX-2 are the pharmacological target of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), including the COX-2 specific inhibitors Vioxx, Bextra, and Celebrex. SPECIFIC AIMS: Using mutagenesis, functional analyses, and x-ray crystallographic methods, we will (1) elucidate the structure of pathogen-inducible oxygenase and characterize at the molecular level the mechanism and structural determinants involved in the stereoselective oxygenation of 18 carbon PUFAs into 2R-oxylipin products; and (2) elucidate the structures of unique 15R-PG producing COX:PUFA complexes in order to understand at the molecular level how the conformation of the PUFA in the active site influences stereospecific oxygenation in the generation of these novel products. HEALTH RELEVANCE: The role that PUFAs play in health and disease is generating renewed interest, with a more focused public perception of healthy food and lifestyle and the significant impact that these compounds have in certain clinical conditions. The ability of proteins, such as COX-2, to dramatically shift their product profiles upon treatment with pharmacological inhibitors has led to further investigations into how these enzymes function. Our studies will provide for a complete mechanistic understanding of how novel lipids are derived from PUFAs upon aspirin treatment, and lend valuable insight into development of new or combined therapeutic approaches for the management of arthritis and vascular inflammation, with fewer unwanted side effects.