Regulators and mediators of TRIM5 function

TRIM5 功能的调节剂和介体

• 批准号: 7991904
• 负责人: Jacek Skowronski
• 金额: $ 17.94万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-11-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991904
• 关键词: Mediator of activation protein

DESCRIPTION (provided by applicant): The overall goal of this application is to understand biochemical mechanisms that underlie the function of rhesus monkey TRIM5 (rhTRIM5). rhTRIM5 blocks HIV-1 infection by acting at two or more post-entry stages. Importantly, rhTRIM5 E3 ubiquitin ligase activity and functional proteasome are important for its full restriction activity. According to the current model of rhTRIM5 function, rhTRIM5 recognizes and binds the incoming restricted virus capsid. This event triggers redistribution of rhTRIM5 molecules to the vicinity of the capsid leading to its accelerated uncoating, and engages the ubiquitin proteasome machinery that mediates capsid degradation. Interestingly, in the cell, rhTRIM5 exhibits a complex behavior pattern characterized by rapid turnover rate and constant exchange of rhTRIM5 molecules between different discrete cytoplasmic compartments. These processes are probably tightly regulated, even in the absence of virus infection, and could be important for rhTRIM5's ability to restrict retrovirus infection. To gain insight into the molecular mechanisms by which rhTRIM5 restricts retrovirus infection, we purified cellular proteins that are bound to rhTRIM5 in intact cells and identified them by a shotgun proteomic approach. Importantly, we found that a significant fraction of rhTRIM5 is specifically associated with 26S proteasome. Thus, we hypothesize that rhTRIM5 binding to 26S proteasome is crucial for the early post-entry infection block to occur. Interestingly, we also identified additional components of ubiquitin proteasome system that specifically associate with rhTRIM5, such as subunits of E3 ubiquitin ligases. These enzymes are likely regulators of rhTRIM5 levels, because rhTRIM5 turnover rate is regulated by polyubiquitylation. In this application we propose to scrutinize the roles of rhTRIM5 binding with 26S proteasome and with other specific components of the ubiquitin proteasome system, for rhTRIM5-mediated restriction and rhTRIM5 turnover rate. Subsequently, we plan to initiate structural studies of selected molecular interactions that are crucial mediators of these processes. Ultimately, our studies will provide a biochemical/molecular framework for understanding the mechanism(s) by which rhTRIM5 blocks HIV-1 infection, and how these mechanism(s) are regulated. This knowledge could lead to the identification of new targets in viral life cycle for development of antivirals and to the conception of new strategies to control HIV infection. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding molecular mechanism that TRIM5 uses to defends cells from infection by HIV-1. Knowledge gained from our studies could lead to the conception of new strategies to control HIV infection and to the identification of new targets for development of anti-retroviral drugs.

描述（由申请人提供）：本申请的总体目标是了解恒河猴 TRIM5 (rhTRIM5) 功能的生化机制。 rhTRIM5 通过在两个或多个进入后阶段发挥作用来阻止 HIV-1 感染。重要的是，rhTRIM5 E3 泛素连接酶活性和功能性蛋白酶体对其完全限制性活性非常重要。根据目前rhTRIM5功能的模型，rhTRIM5识别并结合传入的限制性病毒衣壳。该事件触发 rhTRIM5 分子重新分布到衣壳附近，导致其加速脱壳，并参与介导衣壳降解的泛素蛋白酶体机制。有趣的是，在细胞中，rhTRIM5 表现出复杂的行为模式，其特征是快速周转率和不同离散细胞质区室之间 rhTRIM5 分子的不断交换。即使没有病毒感染，这些过程也可能受到严格调控，并且对于 rhTRIM5 限制逆转录病毒感染的能力可能很重要。为了深入了解 rhTRIM5 限制逆转录病毒感染的分子机制，我们纯化了完整细胞中与 rhTRIM5 结合的细胞蛋白，并通过鸟枪法蛋白质组学方法对其进行了鉴定。重要的是，我们发现 rhTRIM5 的很大一部分与 26S 蛋白酶体特异性相关。因此，我们假设 rhTRIM5 与 26S 蛋白酶体的结合对于早期进入后感染阻断的发生至关重要。有趣的是，我们还鉴定了与 rhTRIM5 特异性相关的泛素蛋白酶体系统的其他成分，例如 E3 泛素连接酶的亚基。这些酶可能是 rhTRIM5 水平的调节因子，因为 rhTRIM5 周转率受多泛素化调节。在此应用中，我们建议仔细研究 rhTRIM5 与 26S 蛋白酶体和泛素蛋白酶体系统其他特定成分结合的作用，以了解 rhTRIM5 介导的限制和 rhTRIM5 周转率。随后，我们计划启动对选定的分子相互作用的结构研究，这些分子相互作用是这些过程的关键介质。最终，我们的研究将为理解 rhTRIM5 阻断 HIV-1 感染的机制以及这些机制是如何调节的提供一个生化/分子框架。这些知识可能有助于确定病毒生命周期中的新目标，以开发抗病毒药物，并制定控制艾滋病毒感染的新策略。 公共健康相关性：拟议的研究旨在了解 TRIM5 用于保护细胞免受 HIV-1 感染的分子机制。从我们的研究中获得的知识可能会导致控制艾滋病毒感染的新策略的构想，并确定抗逆转录病毒药物开发的新靶点。