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，通过泛素蛋白 - 蛋白质途径依赖ATP蛋白质降解可去除对细胞过程的调节至关重要的短期信号蛋白，这对细胞过程至关重要，降解错误折叠和受损的蛋白质，其积累的蛋白质对细胞有毒，对细胞有毒，并破坏异源蛋白以降低抗酸性抗酸性的替代剂。它是理解许多人类疾病的机制，尤其是癌症和神经退行性疾病的基础，例如亨廷顿病。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隔离在其中，两个19S调节颗粒每个都包含6个与20s接触的ATPases。 ATPASE的关键作用是在20年代打开封闭的通道，以促进底物进入破坏。由于19S调节粒子的尺寸较大和动态性质，尽管付出了很大的努力，但整个26S蛋白酶体的结晶仍未成功，并且ATPases控制20S的栅极开放的机制尚待阐明。我们使用替代结构测定技术来阐明这种机制：单个颗粒电子冷冻显微镜（冷冻），该机制不需要蛋白酶体ATPases-20s复合物的结晶。与哈佛医学院的阿尔弗雷德·戈德堡（Alfred Goldberg）教授合作，我们发现ATPASE仅要求其C-Termini诱导开门。因此，我们将ATPase诱导的栅极开放的机械研究与ATP酶的结构测定分开。该应用集中在蛋白酶体ATPase的两个关键问题上：（1）ATPases在20s中如何打开门，以及（2）ATPase循环中ATPases的构象变化。我们的目标是明确定义的，我们的方法是新颖的，独特的，并且被证明是成功的。我们已经通过确定ATPases的C末端诱导20年代的构象变化，从而迈出了关键的一步，从而导致其开口。在AIM 1中，我们将探索控制20年代古细菌变化的决定因素。在AIM 2中，我们将确定真核19S ATPases的C末端是否会引发类似的构象变化，从而导致真核20s的门口开放。在AIM 3中，我们将寻求在其ATPase周期中阐明全长蛋白酶体ATPases的构象变化。这些目标的实质性完成将提高我们对蛋白酶体介导的蛋白质降解的了解，该蛋白质降解在许多人类疾病的发病机理中起着关键作用。它还将推进单个粒子冷冻的技术，以实现更高的分辨率，并检测仅大小的残基的小配体。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分子机降解。蛋白质降解过程受到严格调节，在许多人类疾病的发病机理中起着关键作用，尤其是癌症和神经退行性疾病，例如亨廷顿氏病。该应用研究蛋白酶体ATP酶调节蛋白酶体蛋白水解活性的机制。