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.

项目概要 哺乳动物细胞已经形成了一个复杂的免疫蛋白网络，用于识别和对抗病毒 干扰素刺激基因 15 (ISG15) 是一种 17.2 kDa 串联泛素样蛋白，由特定的 E1– 使用。 最近的生化研究表明，E2-E3 泛素级联酶可干扰病毒蛋白的活性。 证明了 HECT 和含有 RCC1 的蛋白 5 (HERC5) E3 连接酶如何调节 ISG15 信号传导以响应 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。 HERC5 依赖性 ISGylation 的免疫学作用已得到充分证实，HERC5 使用的分子机制 催化 ISG15 特异性且及时地附着于蛋白质以响应病毒感染尚不清楚。 最重要的是，我们了解 HERC5 在原子水平上的工作原理，以帮助未来治疗的开发 治疗病毒感染并增强人类健康。 该项目的目标是了解 HERC5 依赖性 IGSylation 的结构和生化基础。 迄今为止，尚未有关于 HERC5 的结构研究报告，我们将阐明 HERC5 使用的独特机制。 将 ISG15 附着到病毒底物上 HERC5 是 E6AP C 末端 (HECT) E3 的同源成员。 泛素连接酶含有特征性的 HECT 结构域，由 N 端叶和 C 端叶组成， 负责催化 ISG15 与靶蛋白的共价连接，目前这是 HERC5 的机制。 催化 ISG15 与活性位点内部和周围的特定残基的用途和身份 目前尚不清楚 PI 的长期科学目标是研究 HERC5 的 3D 结构和基础酶学。 了解这种酶如何选择性且特异性地将 ISG15 附着到病毒蛋白上，作为宿主先天免疫的一部​​分 回复。 该提案的主要重点是利用结构和生物物理确定 HERC5 的催化机制 方法（目标 1），并检查 HERC5 与 ISG15 和 E2 酶 UBE2L6 的复合物（我们的初步目标 2）。 使用核磁共振波谱和其他生化方法的研究表明，HERC5 的新机制被发现 仅在 HECT 结构域 C 端叶中，其中包含绝对保守的催化半胱氨酸 ISGylate 病毒蛋白建立在我们检查其他成员机制的既定记录的基础上。 HECT E3泛素连接酶，HERC5的固有区别是ISG15特异性，不能催化泛素转移 提供了一个诱人的机会来扩展我们目前对 HECT 依赖性活动的理解以及它们如何 我们的研究结果将为 HECT E3 连接酶使用的分子机制提供新的见解。 HERC5 并帮助我们了解这种酶如何以及为何在应对病毒感染时发挥作用。 R15 AREA 研究项目成功的重要组成部分来自生物化学和分子生物学的贡献。 克拉克大学生物学（BCMB）本科生将成为完成拟议工作的一部分，并且， 由于他们在本研究计划中的重要性，他们将获得 PI 的广泛指导，并将分享 手稿准备和出版作者身份，并在国内和国际研究中展示他们的工作 会议。