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

 描述(申请人提供)：真核细胞已经发展出复杂的防御系统，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，其在抗病毒反应中表达上调的是Viperin(病毒抑制蛋白；内质网相关，干扰素诱导)，它已被证明限制了一些重要的人类病毒的传染性，包括甲型流感、艾滋病毒和丙型肝炎。自由基SAM化学参与哺乳动物的抗病毒反应是完全意想不到的，因为到目前为止研究的自由基SAM酶几乎只涉及微生物代谢。在哺乳动物细胞中的研究表明，许多蛋白质，包括细胞和病毒，都是毒蛇毒素的靶标。然而，这些相互作用都没有被直接表征，毒蛇毒素抑制其靶标酶的机制(S)，包括自由基SAM化学在反应中的作用，仍然不清楚。我们建议研究毒蛇毒素与法尼基焦磷酸合成酶(FPPS)的相互作用，法尼基焦磷酸合成酶是甲氧丙酸生物合成途径中的一个关键酶，也是毒蛇毒素最典型的靶标。根据我们的初步数据和其他自由基SAM酶催化的蛋白质修饰的充分记录，我们假设毒蛇毒素通过共价修饰(例如肽骨架裂解)使FP PS失活，从而导致其降解。该项目的目标是确定毒蛇毒素抑制FPPS的机制，然后将这些研究扩展到其他酶靶标，以确定毒蛇毒素是否通过共同的机制抑制其靶标。这项研究将采取双管齐下的方法，将体外纯化酶的研究与转基因哺乳动物细胞系的免疫标记酶的研究结合起来。体内研究旨在评价蛇毒在生理条件下对FPPS活性的调节作用。靶向蛋白质组学方法将被用来确定毒蛇毒素的其他潜在靶点，并在体内检测毒蛇毒素对FP PS的潜在共价修饰。靶向代谢组学方法将被用来在体内寻找自由基SAM化学的产物，并检测由于抑制FPPS而引起的甲羟戊酸途径中代谢物水平的扰动。体外研究将集中于详细确定毒蛇毒素利用自由基SAM化学来灭活FPPS的机制。根据在FPPS上的实验结果，这些研究将扩展到研究毒蛇毒素与其他靶标酶的相互作用，包括线粒体三功能蛋白，它参与了脂肪酸的氧化分解代谢。

 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.