Structural Biology of Oxylipin Biosynthesis

描述(申请人提供)：目的：本提案的更广泛的目标是了解不同的整膜酶如何利用立体选择性的氧合作用，从一组确定的多不饱和脂肪酸(PUFA)底物中产生独特的氧脂。氧化脂质是由18-22碳多不饱和脂肪酸通过细胞色素P450、脂氧合酶和环氧合酶(COX-1和COX-2)的催化活性通过加成分子氧生物合成的生物活性脂质介体。氧化脂质中最重要的一类是二十烷类化合物，它包括前列腺素(PGs)和从花生四烯酸衍生的白三烯。这些产物负责调节基本的生理过程，并作为炎症过程和其他免疫反应的强有力的脂质介体。环氧合酶-1和环氧合酶-2是阿司匹林和其他非类固醇抗炎药(NSAIDs)的药理靶点，包括环氧合酶-2特异性抑制剂万络、百特和西乐。具体目标：利用诱变、功能分析和X射线结晶学方法，我们将(1)阐明病原体诱导加氧酶的结构，并在分子水平上表征18个碳多不饱和脂肪酸立体选择性氧化生成2R-氧化脂质产物的机制和结构决定因素；以及(2)阐明独特的15R-PG产生COX：PUFA复合体的结构，以便在分子水平上了解活性部位的多不饱和脂肪酸构象如何影响这些新产物的立体选择性氧化作用。与健康相关：多不饱和脂肪酸在健康和疾病中发挥的作用重新引起了人们的兴趣，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重大影响。蛋白质，如COX-2，在用药物抑制剂治疗后显著改变其产物谱的能力导致了对这些酶如何发挥作用的进一步研究。我们的研究将对阿司匹林治疗时如何从多不饱和脂肪酸中提取新的脂质提供一个完整的机制理解，并为开发新的或联合治疗方法来管理关节炎和血管炎症提供有价值的见解，同时减少不良副作用。

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.