Structure and Mechanism of HERC5-dependent ISGylation

HERC5 依赖性 ISGylation 的结构和机制

• 批准号: 10792678
• 负责人: Donald Eric Spratt
• 金额: $ 9.8万
• 依托单位: CLARK UNIVERSITY (WORCESTER, MA)
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10792678
• 关键词: 2019-nCoV; 8 year old; Active Sites; Authorship; Biochemical; Biochemistry; C-terminal; Characteristics; Complex; Cysteine; Development; Disease; Enzymatic Biochemistry; Enzymes; Equipment; Functional disorder; Future; Genes; Goals; HIV; Health; Hepatitis C; Human; Immunologics; Influenza; Innate Immune Response; Interferons; International; Learning; Lobe; Mammalian Cell; Manuscripts; Molecular; Molecular Biology; N-terminal; NMR Spectroscopy; Preparation; Proteins; Publications; Reporting; Research; Research Project Grants; Role; Signal Transduction; Structure; System; UBE3A gene; Ubiquitin; Ubiquitin Like Proteins; Universities; Viral; Viral Physiology; Viral Proteins; Virus Diseases; Work; biophysical techniques; combat; fast protein liquid chromatography; improved; insight; member; novel; pathogenic virus; programs; response; success; symposium; therapy development; three dimensional structure; ubiquitin-protein ligase; undergraduate student

Project Summary Mammalian cells have developed an elaborate network of immunoproteins that serve to identify and combat viral pathogens. Interferon-stimulated gene 15 (ISG15) is a 17.2 kDa tandem ubiquitin-like protein that is used by specific E1– E2–E3 ubiquitin cascade enzymes to interfere with the activity of viral proteins. Recent biochemical studies have demonstrated how the HECT and RCC1-containing protein 5 (HERC5) E3 ligase regulates ISG15 signaling in response to SARS-CoV-2, hepatitis C (HCV), influenza-A (IAV), human immunodeficiency virus (HIV), and other viral infections. While the immunological role of HERC5-dependent ISGylation is well established, the molecular mechanisms used by HERC5 to catalyze the specific and timely attachment of ISG15 to proteins in response to a viral infection remain unclear. It is paramount that we understand how HERC5 works at the atomic level to aid in the future development of therapies to treat viral infections and to enhance human health. The objective of this project is to understand the structural and biochemical basis for HERC5-dependent IGSylation. To date there have been no structural studies reported for HERC5. We will elucidate the unique mechanism used by HERC5 to attach ISG15 on to viral substrates. HERC5 is a unique member of the Homologous to E6AP C-Terminus (HECT) E3 ubiquitin ligases that contains the characteristic HECT domain, consisting of an N-terminal lobe and a C-terminal lobe, that is responsible for catalyzing the covalent attachment of ISG15 to a target protein. Currently the mechanism that HERC5 uses and the identities of specific residues in and around the active site required to catalyze the attachment of ISG15 with is unclear. The long-term scientific goal of the PI is to investigate the 3D structures and underlying enzymology for HERC5 to learn how this enzyme selectively and specifically attaches ISG15 to viral proteins as part of the host’s innate immune response. The major foci of this proposal will be to determine the catalytic mechanism of HERC5 using structural and biophysical approaches (Aim 1), and to examine HERC5 complex with ISG15 and the E2 enzyme UBE2L6 (Aim 2). Our preliminary studies using NMR spectroscopy and other biochemical approaches suggest that the novel mechanism of HERC5 is found exclusively in the HECT domain C-terminal lobe that contain the absolutely conserved catalytic cysteine required to ISGylate viral proteins. Building on our established track-record of examining the mechanisms of other members of the HECT E3 ubiquitin ligases, the inherent difference of HERC5 being ISG15-specific and not able to catalyze ubiquitin transfer provide an enticing opportunity to expand our current understanding of HECT-dependent activity and how their dysfunctions cause disease. Our findings will offer new insight into the molecular mechanisms used by the HECT E3 ligase HERC5 and help us learn how and why this enzyme works in response to a viral infection. Undergraduate students will be an essential part of the success of this R15 AREA research project. The contribution from Biochemistry and Molecular Biology (BCMB) undergraduate students at Clark University will be integral to the completion of the proposed work and, as a result of their importance in this research program, they will receive extensive guidance from the PI, will share in manuscript preparation and publication authorship, and present their work at national and international research conferences.

项目摘要 哺乳动物细胞已经发展出一个复杂的免疫蛋白网络， 病原体干扰素刺激基因15（Interferon-stimulated gene 15，ISG 15）是一个17.2 kDa的串联泛素样蛋白，被特异性E1- E2-E3泛素级联酶干扰病毒蛋白的活性。最近的生物化学研究 证明了HECT和含RCC 1的蛋白5（HERC 5）E3连接酶如何调节ISG 15信号转导， SARS-CoV-2、丙型肝炎（HCV）、甲型流感（IAV）、人类免疫缺陷病毒（HIV）和其他病毒感染。而 HERC 5依赖性ISG化的免疫学作用已被充分确立，HERC 5用于 催化ISG 15特异性和及时地附着到蛋白质上以响应病毒感染仍然不清楚。是 最重要的是，我们了解HERC 5如何在原子水平上工作，以帮助未来的治疗发展， 治疗病毒感染和增进人类健康。 该项目的目的是了解HERC 5依赖性IGS化的结构和生化基础。到 迄今为止，尚未报道HERC 5的结构研究。我们将阐明HERC 5的独特机制， 将ISG 15附着在病毒基质上。HERC 5是E6 AP C-末端同源序列（HECT）E3的独特成员 泛素连接酶，包含特征性HECT结构域，由N-末端叶和C-末端叶组成， 负责催化ISG 15与靶蛋白的共价连接。目前，HERC 5 催化ISG 15与 还不清楚PI的长期科学目标是研究HERC 5的3D结构和基础酶学 为了了解这种酶如何选择性地和特异性地将ISG 15附着到病毒蛋白上，作为宿主先天免疫的一部分， 反应 该提案的主要重点是利用结构和生物物理确定HERC 5的催化机制 方法（目的1），并检查HERC 5与ISG 15和E2酶UBE 2L 6的复合物（目的2）。我们的初步 使用NMR光谱和其他生物化学方法的研究表明，发现了HERC 5的新机制， 仅在HECT结构域C-末端叶中，其含有绝对保守的催化半胱氨酸， ISGylate病毒蛋白。在我们审查联合国其他成员机制的既定记录的基础上， HECT E3泛素连接酶，HERC 5的固有差异是ISG 15特异性的，不能催化泛素转移 提供了一个诱人的机会，以扩大我们目前的理解HECT依赖的活动，以及他们如何 功能障碍导致疾病。我们的发现将为HECT E3连接酶的分子机制提供新的见解 HERC 5并帮助我们了解这种酶如何以及为什么在应对病毒感染时起作用。本科生将在 这是R15区域研究项目成功的重要组成部分。生物化学与分子生物学的贡献 生物学（BCMB）本科生在克拉克大学将是不可或缺的完成拟议的工作， 由于他们在这项研究计划中的重要性，他们将得到PI的广泛指导，将分享 手稿准备和出版物的作者，并提出他们的工作在国家和国际研究 两会