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（由适用提供）：真核细胞已经开发出旨在限制病毒复制的复杂防御措施，从而防止感染升级为其他细胞。 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 infection of a number of important human viruses include influenceza 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 家庭;这些酶还原以产生一种对催化至关重要的腺苷自由基。激进的SAM化学参与哺乳动物抗病毒反应是完全出乎意料的，因为到目前为止，我们几乎完全提议研究耐二烯基焦磷酸合酶（FPPS）的相互作用，这是一种元素酸酯式途径的关键酶（FPPS），该目标的目标是最佳量和库存量。基于我们的初步数据和据可记录的蛋白质修饰，由其他激进的SAM酶催化，我们假设耐蛋白通过共价修饰使FPP失活，例如Pepperonate-Backbone裂解，导致其降解。该项目的目标是确定耐二素抑制FPP的机制，然后将这些研究扩展到其他酶靶标，以确定耐二素是否通过共同的机制抑制其靶标。这项研究将采用两种普通的方法，将体外纯化酶与对哺乳动物细胞系中翻译的免疫标记酶的研究相结合。体内研究旨在评估在物理条件下通过耐比蛋白对FPPS活性的调节。靶向蛋白质组学方法将用于识别耐蛋白蛋白的其他潜在靶标，并检测Viperin在体内对FPP的潜在共价修饰。靶向代谢组学方法将用于搜索体内激进的SAM化学产品的产物，并检查由FPP抑制引起的Mevalonate途径中代谢物水平的扰动。体外研究将侧重于详细确定耐蛋白利用激进的SAM化学灭活FPP的机制。通过在FPP上的实验结果的情况下，将扩展研究以检查耐二蛋白与其他靶酶的相互作用，包括线粒体三官能蛋白，该蛋白与β-氧化途径有关脂肪酸的分解代谢。