Targets and mechanism of a radical SAM enzyme involved in the cellular antiviral response

参与细胞抗病毒反应的自由基 SAM 酶的靶标和机制

• 批准号: 8960243
• 负责人: E NEIL MARSH
• 金额: $ 29.28万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8960243
• 关键词: 5' -deoxyadenosine; Animals; Antiviral Agents; Antiviral Response; Binding; Biochemical; C-terminal; Catabolism; Catalysis; Cell Line; Cells; Chemistry; Cholesterol; Cleaved cell; Complex; Crystallization; Data; Endoplasmic Reticulum; Enzymes; Epitopes; Eukaryotic Cell; Family; Fatty Acids; Gene Expression Regulation; Genes; Goals; HIV; Hepatitis C; Human; Human Virus; In Vitro; Infection; Infection prevention; Influenza; Influenza A virus; Interferons; Investigation; Laboratories; Lead; Lipids; Mammalian Cell; Mediating; Metabolism; Mitochondria; Modification; Multienzyme Complexes; Pathway interactions; Peptides; Physiological; Play; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reaction; Regulation; Research; Role; S-Adenosylmethionine; System; Vertebral column; Viral; Virus; Virus Diseases; base; cholesterol biosynthesis; effective therapy; experience; farnesyl pyrophosphate; fatty acid oxidation; flexibility; in vivo; insight; interest; member; metabolomics; mevalonate; microbial; oxidation; pathogen; prevent; public health relevance; research study; viperin

 DESCRIPTION (provided by applicant): Eukaryotic cells have developed sophisticated defenses aimed at limiting viral replication and thereby preventing infection from escalating to other cells. Among the many interferon-stimulated genes whose expression is up-regulated in the antiviral response is viperin (Virus Inhibitory Protein; Endoplasmic Reticulum associated, INterferon inducible), which has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV and hepatitis C. One of the most interesting features of viperin is that it appears to be a member of the radical SAM enzyme family; these enzymes reductively cleave S- adenosylmethionine to generate an adenosyl radical that is essential for catalysis. The involvement of radical SAM chemistry in the mammalian antiviral response was completely unexpected, as the radical SAM enzymes so far studied have almost exclusively been involved in microbial metabolism. Studies in mammalian cells have implicated a number of proteins, both cellular and viral, as targets of viperin. However, none of these interactions have been characterized directly and the mechanism(s) by which viperin inhibits its target enzymes, including the role of radical SAM chemistry in the reaction, remain unknown. We propose to study the interaction of viperin with farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate biosynthetic pathway and the best-characterized target of viperin. Based on our preliminary data and well-documented protein modifications catalyzed by other radical SAM enzymes, we hypothesize that viperin inactivates FPPS by covalent modification, e.g. peptide-backbone cleavage, leading to its degradation. The project's goals are to determine the mechanism by which viperin inhibits FPPS, and then to extend these studies to other enzyme targets to establish whether viperin inhibits its targets by a common mechanism. The research will take a two-pronged approach that will combine studies on purified enzymes in vitro with studies on immuno-tagged enzymes transfected in mammalian cell lines. In vivo studies aim to evaluate the regulation of FPPS activity by viperin under physiological conditions. Targeted proteomics approaches will be used to identify other potential targets of viperin and to detect potential covalent modifications of FPPS by viperin in vivo. Targeted metabolomics approaches will be used to search for the products of radical SAM chemistry in vivo and to examine the perturbation of metabolites levels in the mevalonate pathway arising from inhibition of FPPS. In vitro studies will focus on determining in detail the mechanism by which viperin harnesses radical SAM chemistry to inactivate FPPS. Informed by the results of experiments on FPPS, the studies will be extended to examine the interaction of viperin with other target enzymes, including the mitochondrial trifunctional protein, which is involved in the catabolism of fatty acids by the ß-oxidation pathway.

 描述（由申请人提供）：真核细胞已经发展出复杂的防御机制，旨在限制病毒复制，从而防止感染升级到其他细胞。在许多干扰素刺激的基因中，其表达在抗病毒应答中上调的是viperin（病毒抑制蛋白;内质网相关，干扰素诱导型），其已显示限制许多重要的人类病毒（包括甲型流感、HIV和丙型肝炎）的感染性。蝰蛇蛋白最有趣的特征之一是它似乎是自由基SAM酶家族的成员;这些酶还原性切割S-腺苷甲硫氨酸以产生催化所必需的腺苷自由基。自由基SAM化学在哺乳动物抗病毒反应中的参与是完全出乎意料的，因为迄今为止研究的自由基SAM酶几乎完全参与微生物代谢。 在哺乳动物细胞中的研究已经涉及许多蛋白质，包括细胞和病毒，作为蝰蛇蛋白的靶标。然而，这些相互作用中没有一个被直接表征，并且蝰蛇蛋白抑制其靶酶的机制（包括自由基SAM化学在反应中的作用）仍然未知。 我们建议研究蝰蛇蛋白与法尼基焦磷酸合酶（FPPS），甲羟戊酸生物合成途径中的关键酶和蝰蛇蛋白的最佳特征的目标的相互作用。基于我们的初步数据和充分记录的蛋白质修饰催化的其他自由基SAM酶，我们假设蝰蛇蛋白灭活FPPS的共价修饰，例如肽骨架切割，导致其降解。该项目的目标是确定蝰蛇蛋白抑制FPPS的机制，然后将这些研究扩展到其他酶靶点，以确定蝰蛇蛋白是否通过共同机制抑制其靶点。 这项研究将采取双管齐下的方法，将联合收割机对体外纯化酶的研究与对哺乳动物细胞系中转染的免疫标记酶的研究相结合。体内研究旨在评估在生理条件下蝰蛇蛋白对FPPS活性的调节。靶向蛋白质组学方法将用于鉴定蝰蛇蛋白的其他潜在靶点，并检测体内蝰蛇蛋白对FPPS的潜在共价修饰。有针对性的代谢组学方法将用于搜索自由基SAM化学在体内的产品，并检查由FPPS抑制引起的甲羟戊酸途径中的代谢物水平的扰动。体外研究将集中于详细确定蝰蛇素利用自由基SAM化学来抑制FPPS的机制。根据FPPS实验的结果，这些研究将扩展到检查蝰蛇蛋白与其他靶酶的相互作用，包括线粒体三功能蛋白，其通过β-氧化途径参与脂肪酸的催化。