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.

通过共价附着泛素或泛素样修饰剂的共价附着，对靶蛋白的翻译后修饰是细胞信号传导的关键机制。泛素介导的某些蛋白质因子通过靶向26S蛋白酶体的去除的核心生物学作用越来越清楚。此外，泛素信号传导的缺陷直接与越来越多的疾病有关，例如帕金森氏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分析对于理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并在患病状态下有缺陷至关重要。通过由激活（E1），共轭（E2）和连接酶（E3）酶组成的酶级联酶来实现泛素蛋白的添加。多泛素化还可能需要一种称为E4连接酶的专用多泛素连接酶。我们的研究集中于新发现的E3连接酶的U-box域家族，这些域刚刚开始表征。我们的实验室从必需的RNA剪接因子PRP19确定了U盒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AIM 1中，我们建议扩展这些研究以生成完整的U-box连接酶的完整结构模型，目前尚无示例。 AIM 2专注于U-box家族的其他两个成员ChIP和UFD2，仅具有E3活性，但彼此绑定时会表现出独特的E4聚泛素化活性。该目标的目的是确定E3与E4活性的结构和功能基础。 CHIP具有出色的能力，可以在其作为HSP70的副伴侣中的两个不同功能之间切换，并用作E3连接酶或促进蛋白质重折叠。因此，芯片似乎是响应细胞应激的重折叠和脱发状态之间切换的关键因素。 AIM 3致力于发展对这种切换机制的理解。我们提出了芯片相互作用的生物化学和结构表征：（i）ubc5，其互补的E2连接酶，（ii）Bag-1，刺激芯片作为泛素链氨基链蛋白 - 蛋白 - 蛋白的活性，以及​​（iii）hspbpi，竞争性的co-chaperone，其切换蛋白质的蛋白质。