Structure and Mechanism of HERC5-dependent ISGylation

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，用于识别和对抗病毒。 病原体。干扰素刺激基因15(ISG15)是一种17.2 kDa的串联泛素样蛋白，由特异性的E1- E2-E3泛素级联酶干扰病毒蛋白的活性。最近的生化研究表明 演示了Hect和包含RCC1的蛋白5(HERC5)E3连接酶如何调节ISG15信号转导 SARS-CoV-2、丙型肝炎(丙型肝炎)、甲型流感(IAV)、人类免疫缺陷病毒(HIV)和其他病毒感染。而当 HERC5依赖的ISG化在免疫学中的作用已经得到了很好的证实，HERC5用来 对于病毒感染的反应，催化ISG15特异和及时地附着在蛋白质上的作用尚不清楚。它是 最重要的是，我们了解HERC5如何在原子水平上工作，以帮助未来的治疗方法的发展 治疗病毒感染，增进人类健康。 本项目的目的是了解HERC5依赖的IGSylation的结构和生化基础。至 目前还没有关于HERC5的结构研究的报道。我们将阐明HERC5使用的独特机制 将ISG15附着在病毒底物上。HERC5是E6AP C-末端(Hect)E3的唯一同源成员 泛素连接酶，含有特征的Hect结构域，由N-末端叶和C-末端叶组成， 负责催化ISG15与靶蛋白的共价结合。目前，HERC5的机制是 催化ISG15与ISG15结合所需的活性中心及其周围特定残基的用途和特性 目前还不清楚。PI的长期科学目标是研究HERC5的3D结构和潜在的酶学 为了了解这种酶如何选择性和特异性地将ISG15连接到病毒蛋白上，作为宿主先天免疫的一部分 回应。 这项提议的主要焦点将是利用结构和生物物理来确定HERC5的催化机制。 方法(目标1)，并检测HERC5与ISG15和E2酶UBE2L6的复合体(目标2)。我们的预赛 核磁共振波谱和其他生化方法的研究表明，发现了HERC5的新机制 仅在Hect结构域的C-末端叶中，含有绝对保守的催化半胱氨酸，需要 ISGylate病毒蛋白。在我们审查本组织其他成员机制的既定记录的基础上 在E3泛素连接酶中，HERC5的固有差异是ISG15特异的，不能催化泛素转移 提供了一个诱人的机会来扩大我们目前对Hect依赖活动的理解，以及他们如何 功能障碍会导致疾病。我们的发现将为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什么在应对病毒感染时起作用。本科生将成为 这是R15地区研究项目成功的关键部分。生物化学和分子科学的贡献 克拉克大学的生物学(BCMB)本科生将是完成拟议工作不可或缺的一部分， 由于他们在本研究计划中的重要性，他们将从PI那里获得广泛的指导，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。

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.