Structure and Mechanism of HERC5-dependent ISGylation

HERC5 依赖性 ISGylation 的结构和机制

• 批准号: 10439061
• 负责人: Donald Eric Spratt
• 金额: $ 45.09万
• 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/10439061
• 关键词: 2019-nCoV; 3-Dimensional; Active Sites; Antiviral Agents; Antiviral Response; Authorship; Biochemical; Biochemistry; Biological Assay; Biophysics; C-terminal; Calorimetry; Cell membrane; Cells; Characteristics; Charge; Chemicals; Chemistry; Complex; Coupled; Cysteine; Development; Disease; Drug Targeting; Ebola virus; Educational process of instructing; Environment; Enzymatic Biochemistry; Enzymes; Functional disorder; Future; Genes; Goals; HIV; Health; Hepatitis C; Heteronuclear NMR; Human; Immunologics; Influenza; Influenza A virus; Innate Immune Response; Interferons; International; Knowledge; Learning; Link; Lobe; Mammalian Cell; Manuscripts; Mediating; Molecular; Molecular Biology; Monitor; N-terminal; NMR Spectroscopy; Nuclear Magnetic Resonance; Pharmaceutical Preparations; Pharmacotherapy; Play; Preparation; Process; Production; Protein Biosynthesis; Proteins; Publications; Reporting; Research; Research Project Grants; Role; SARS-CoV-2 antiviral; Scientific Advances and Accomplishments; Signal Transduction; Site-Directed Mutagenesis; Structure; Testing; Time; Titrations; Training; UBE3A gene; Ubiquitin; Ubiquitin Like Proteins; Universities; Viral; Viral Pathogenesis; Viral Physiology; Viral Proteins; Virion; Virus Diseases; Virus Replication; Work; X-Ray Crystallography; antiviral immunity; biophysical techniques; combat; experimental study; human disease; improved; innovation; 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 哺乳动物细胞已经建立了一个精致的免疫蛋白网络，该网络用于识别和对抗病毒 病原体。干扰素刺激的基因15（ISG15）是17.2 kDa串联泛素样蛋白，特定E1 –使用 E2 – E3泛素级联酶干扰病毒蛋白的活性。最近的生化研究已经 证明了HECT和含RCC1的蛋白5（HERC5）E3连接酶如何调节ISG15信号的响应 SARS-COV-2，丙型肝炎（HCV），影响力-A（IAV），人免疫缺陷病毒（HIV）和其他病毒感染。尽管 HERC5依赖性iSgylation的免疫学作用已经确定，HERC5使用的分子机制 催化ISG15响应病毒感染的特异性和及时附着在蛋白质上，尚不清楚。这是 我们了解HERC5在原子层如何工作以帮助未来的疗法开发到的最重要的 治疗病毒感染并增强人类健康。 该项目的目的是了解HERC5依赖性igsylation的结构和生化基础。到 日期没有针对HERC5报告的结构研究。我们将阐明HERC5使用的独特机制 将ISG15连接到病毒底物上。 HERC5是E6AP C末端（HECT）E3同源的独特成员 泛素连接酶包含特征性Hect域，由N末端叶和C末端叶组成 负责催化ISG15与靶蛋白的共价附着。目前HERC5的机制 用途和特定残留物的身份在催化ISG15附着的活性位点和周围 不清楚。 PI的长期科学目标是研究HERC5的3D结构和基础酶学 为了了解该酶如何选择性地将ISG15连接到病毒蛋白上，作为宿主先天免疫的一部​​分 回复。 该提案的主要焦点是使用结构和生物物理学来确定HERC5的催化机制 接近（目标1），并与ISG15和E2酶UBE2L6（AIM 2）一起检查HERC5复合物。我们的初步 使用NMR光谱和其他生化方法的研究表明，发现HERC5的新机制 仅在含有绝对保守的催化性半胱氨酸的hect域C末端叶中 Isgylate病毒蛋白。在我们既定的轨道上建立了研究其他成员的机制 HECT E3泛素连接酶，HERC5的继承差异为ISG15特异性，无法催化泛素转移 提供了一个诱人的机会，以扩大我们当前对依赖公交活动的理解以及如何 功能障碍引起疾病。我们的发现将提供有关HECT E3连接酶使用的分子机制的新见解 HERC5并帮助我们了解这种酶如何以及为什么响应病毒感染作用。本科生将 该R15地区研究项目成功的重要组成部分。生物化学和分子的贡献 克拉克大学的生物学（BCMB）本科生将是拟议工作的完成不可或缺的一部分 由于他们在该研究计划中的重要性，他们将获得PI的广泛指导，将分享 手稿准备和出版作者身份，并在国家和国际研究中介绍他们的作品 会议。