Mechanism of gate-opening in the 20S proteasome induced by the proteasomal ATPase

蛋白酶体ATP酶诱导20S蛋白酶体开门的机制

• 批准号: 7750590
• 负责人: Yifan Cheng
• 金额: $ 28.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750590
• 关键词: 26S proteasome; ATP phosphohydrolase; Active Sites; Cell physiology; Cells; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Eukaryota; Eukaryotic Cell; Huntington Disease; Immune system; Knowledge; Lead; Length; Life; Ligands; Malignant Neoplasms; Mediating; Molecular Machines; Names; Nature; Neurodegenerative Disorders; Pathogenesis; Pathway interactions; Peptides; Play; Principal Investigator; Process; Proteins; Regulation; Research; Resolution; Role; Signaling Protein; Structure; Techniques; Technology; Ubiquitin; human disease; medical schools; multicatalytic endopeptidase complex; novel; particle; professor; programs; protein degradation

DESCRIPTION (provided by applicant): In eukaryotes the ATP dependent protein degradation by the ubiquitin-proteasome pathway removes short lived signaling protein that is critical in regulation of cellular process, degrades misfolded and damaged proteins whose accumulation is toxic to the cell and breaks down foreign proteins to generate antigenic peptides for presenting to the immune system. It is fundamental in understanding the mechanism of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington disease. The eukaryotic 26S proteasome is formed by a 20S proteasome with the proteolytic active sites sequestered inside it and two 19S regulatory particles each contain six ATPases in contact with the 20S. A key role of the ATPases is to open the gated channel in the 20S to facilitate substrates enter for destruction. Because of the large size and dynamic nature of the 19S regulatory particle, crystallization of the entire 26S proteasome for structure determination remains unsuccessful despite substantial efforts, and the mechanism by which the ATPases controls the gate-opening in the 20S remains to be elucidated. We use an alternative structure determination technique to elucidate this mechanism: single particle electron cryomicroscopy (cryoEM) which does not require crystallization of proteasomal ATPases-20S complex. In collaboration with Professor Alfred Goldberg from Harvard Medical School, we have found that the ATPases only require their C-termini to induce the gate-opening. We thus separated the mechanistic studies of ATPase induced gate-opening from the structure determination of the ATPases. This application focuses on two critical issues of the proteasomal ATPases: (1) how the ATPases opens the gate in 20S and (2) the conformational changes of ATPases during the ATPase cycle. Our aims are clearly defined and our approach is novel, unique and has been proven successful. We already made a critical step forward by determining that the C-termini of ATPases induce a conformational change in the archaeal 20S that leads to its gate-opening. In Aim 1 we will explore the determinants that govern such conformational changes in archaeal 20S. In Aim 2, we will determine if the C-termini of eukaryotic 19S ATPases trigger similar conformational changes that lead to gate-opening in the eukaryotic 20S. In Aim 3 we will seek to elucidate the conformational changes of full length proteasomal ATPases during its ATPase cycle. Substantial completion of these aims will advance our knowledge about the proteasome-mediated protein degradation that plays a key role in the pathogenesis of many human diseases. It will also advance the technology of single particle cryoEM to achieve higher resolutions and to detect small ligand that is only a few residues in size. In eukaryotic cells most unwanted proteins are degraded by a large molecular machine named proteasome. The protein degradation process is tightly regulated and plays a key role in the pathogenesis of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington's disease. This application studies the mechanism by which the proteasomal ATPases regulate the proteolytic activities of the proteasome.

描述（由申请人提供）：在真核生物中，泛素-蛋白酶体途径的 ATP 依赖性蛋白质降解去除了在细胞过程调节中至关重要的短命信号蛋白，降解了错误折叠和受损的蛋白质，这些蛋白质的积累对细胞有毒，并分解了外源蛋白。蛋白质产生抗原肽以呈递给免疫系统。它对于理解许多人类疾病的机制至关重要，特别是癌症和神经退行性疾病，例如神经退行性疾病。亨廷顿病。真核26S蛋白酶体由20S蛋白酶体形成，其中蛋白水解活性位点被隔离在其中，两个19S调节颗粒各自含有六个与20S接触的ATP酶。 ATP 酶的一个关键作用是在 20 年代打开门控通道，以促进底物进入并被破坏。由于 19S 调节颗粒的大尺寸和动态性质，尽管付出了大量努力，用于结构测定的整个 26S 蛋白酶体的结晶仍然不成功，并且 ATP 酶控制 20S 中大门打开的机制仍有待阐明。我们使用另一种结构测定技术来阐明这一机制：单粒子电子冷冻显微镜 (cryoEM)，它不需要蛋白酶体 ATPases-20S 复合物的结晶。我们与哈佛医学院的 Alfred Goldberg 教授合作，发现 ATPase 仅需要其 C 末端来诱导门打开。因此，我们将 ATP 酶诱导的开门机制研究与 ATP 酶的结构测定分开。该应用重点关注蛋白酶体 ATP 酶的两个关键问题：(1) ATP 酶如何在 20 秒内打开大门；(2) ATP 酶循环期间 ATP 酶的构象变化。我们的目标明确，我们的方法新颖、独特，并已被证明是成功的。我们已经向前迈出了关键的一步，确定 ATP 酶的 C 末端会诱导古菌 20S 发生构象变化，从而导致其大门打开。在目标 1 中，我们将探索控制古菌 20S 构象变化的决定因素。在目标 2 中，我们将确定真核 19S ATP 酶的 C 末端是否会触发类似的构象变化，从而导致真核 20S 中的大门打开。在目标 3 中，我们将寻求阐明全长蛋白酶体 ATP 酶在 ATP 酶循环过程中的构象变化。这些目标的实质性完成将增进我们对蛋白酶体介导的蛋白质降解的了解，这种降解在许多人类疾病的发病机制中发挥着关键作用。它还将推进单粒子冷冻电镜技术，以实现更高的分辨率并检测只有几个残基大小的小配体。在真核细胞中，大多数不需要的蛋白质被称为蛋白酶体的大型分子机器降解。蛋白质降解过程受到严格调控，在许多人类疾病的发病机制中发挥着关键作用，特别是癌症和神经退行性疾病，例如癌症。亨廷顿病。该应用研究了蛋白酶体 ATP 酶调节蛋白酶体的蛋白水解活性的机制。