Structural Biology of U-box E3 Ubiquitin Ligases

U-box E3 泛素连接酶的结构生物学

• 批准号: 6964576
• 负责人: WALTER J. CHAZIN
• 金额: $ 26.59万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-10 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6964576
• 关键词: X ray crystallography; binding proteins; biological signal transduction; chemical addition; covalent bond; electron microscopy; enzyme activity; enzyme mechanism; heat shock proteins; ligase; model design /development; molecular chaperones; nuclear magnetic resonance spectroscopy; physical model; posttranslational modifications; protein degradation; protein folding; protein protein interaction; protein structure function; structural biology; ubiquitin

Post-translational modification of target proteins via covalent attachment of ubiquitin or ubiquitin-like modifiers is a key mechanism for cellular signaling. The central biological role of ubiquitin mediated removal of certain protein factors through targeting to the 26S proteasome has become increasingly clear. Moreover, defects in ubiquitin signaling are directly implicated in an increasing number of diseases, such as Parkinson's and other neurodegenerative disorders as well as a variety of human cancers, such as breast and ovarian cancer as well as various types of lymphoma. Coupled structural and functional analysis of the ubiquitination process is essential to understanding how this common signaling process modulates protein function in healthy tissue and is defective in diseased states. Addition of ubiquitin to target proteins is accomplished via an enzyme cascade consisting of activating (E1), conjugating (E2), and ligating (E3) enzymes. Poly-ubiquitination also can require a specialized poly-ubiquitin ligase, termed an E4 ligase. Our studies focus on the newly discovered U-box domain family of E3 ligases, which are just beginning to be characterized. Our laboratory determined the first structure of a U-box domain from an essential RNA splicing factor, Prp19, and showed that the U-box was required for E3 ligase activity. In Aim 1, we propose extending these studies to generate a complete structural model of the intact U-box ligase, for which there are currently no examples. Aim 2 focuses on two other members of the U-box family, CHIP and Ufd2, which alone have E3 activity but when bound to each other exhibit the unique E4 poly-ubiquitination activity. The goal of this Aim is to determine the structural and functional basis for E3 versus E4 activity. CHIP has the remarkable ability to switch between two different functions in its role as a co-chaperone of Hsp70, serving as either an E3 ligase or promoting protein refolding. Thus, CHIP appears to serve as a key factor enabling switching between refolding and degratory regimes in response to cellular stress. Aim 3 is directed towards developing an understanding of this switching mechanism. We propose biochemical and structural characterization of the interactions of CHIP with: (i) Ubc5, its complementary E2 ligase, (ii) Bag-1, the co-chaperone that stimulates CHIP'S activity as a ubiquitin ligase, and (iii) HspBPI, the competing co-chaperone that switches CHIP to its protein refolding regime.

通过共价连接泛素或泛素样修饰物对靶蛋白进行翻译后修饰是细胞信号传导的关键机制。泛素通过靶向 26S 蛋白酶体介导去除某些蛋白质因子的核心生物学作用已变得越来越清晰。此外，泛素信号传导缺陷与越来越多的疾病直接相关，例如帕金森病和其他神经退行性疾病以及各种人类癌症，例如乳腺癌和卵巢癌以及各种类型的淋巴瘤。泛素化过程的结构和功能耦合分析对于了解这种常见信号传导过程如何调节健康组织中的蛋白质功能以及在疾病状态下存在缺陷至关重要。将泛素添加到靶蛋白上是通过由激活酶 (E1)、缀合酶 (E2) 和连接酶 (E3) 组成的酶级联完成的。多聚泛素化还需要专门的多聚泛素连接酶，称为 E4 连接酶。我们的研究重点是新发现的 E3 连接酶 U-box 结构域家族，该家族刚刚开始被表征。我们的实验室从必需的 RNA 剪接因子 Prp19 中确定了 U-box 结构域的第一个结构，并表明 U-box 是 E3 连接酶活性所必需的。在目标 1 中，我们建议扩展这些研究以生成完整 U 型盒连接酶的完整结构模型，但目前尚无示例。目标 2 重点关注 U-box 家族的另外两个成员 CHIP 和 Ufd2，它们单独具有 E3 活性，但当彼此结合时表现出独特的 E4 多聚泛素化活性。该目标的目标是确定 E3 与 E4 活性的结构和功能基础。 CHIP 作为 Hsp70 的共伴侣，具有在两种不同功能之间切换的卓越能力，既可以充当 E3 连接酶，也可以促进蛋白质重折叠。因此，CHIP 似乎是响应细胞应激而在重折叠和降解机制之间切换的关键因素。目标 3 旨在加深对这种切换机制的理解。我们提出了 CHIP 与以下相互作用的生化和结构表征：(i) Ubc5，其互补 E2 连接酶；(ii) Bag-1，作为泛素连接酶刺激 CHIP 活性的辅助伴侣；(iii) HspBPI，将 CHIP 切换到其蛋白质重折叠机制的竞争性共伴侣。