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，其中viperin（病毒抑制基因）的表达在抗病毒反应中上调。蛋白质；内质网相关，干扰素诱导），已被证明可以限制许多重要的人类病毒的感染性，包括甲型流感病毒、艾滋病毒和丙型肝炎。蝰蛇蛋白最有趣的特征之一是它似乎是自由基 SAM 酶家族的一员；这些酶还原性裂解 S-腺苷甲硫氨酸，生成对于催化自由基 SAM 化学至关重要的腺苷自由基。哺乳动物抗病毒反应中的这种变化是完全出乎意料的，因为迄今为止研究的自由基 SAM 酶几乎完全涉及微生物代谢。然而，这些相互作用都没有被直接表征，并且 viperin 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。根据我们的初步数据和结果，我们建议研究 viperin 与法呢基焦磷酸合酶 (FPPS) 的相互作用，FPPS 是甲羟戊酸生物合成途径中的关键酶，也是 viperin 的最佳表征靶点。通过其他自由基 SAM 酶催化的蛋白质修饰，我们研究了 viperin 通过共价修饰（例如肽骨架裂解）使 FPPS 失活，从而导致其降解。该项目的目标是确定 viperin 抑制 FPPS 的机制，然后研究 viperin 的作用。将这些研究扩展到其他酶靶标，以确定 viperin 是否通过共同机制抑制其靶标。该研究将采取双管齐下的方法，将纯化的研究结合起来。体外酶和在哺乳动物细胞系中转染的免疫标记酶的研究旨在评估生理条件下 viperin 对 FPPS 活性的调节，将用于识别 viperin 的其他潜在靶点并检测潜在的靶点。 viperin 体内对 FPPS 的共价修饰将用于寻找体内自由基 SAM 化学的产物并检查体内代谢物水平的扰动。体外研究将集中于详细确定 viperin 利用自由基 SAM 化学使 FPPS 失活的机制。根据 FPPS 的实验结果，这些研究将扩展到检查 viperin 的相互作用。与其他靶酶，包括线粒体三功能蛋白，它通过 β-氧化途径参与脂肪酸的分解代谢。