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

描述（由申请人提供）：在真核生物中，通过泛素-蛋白酶体途径进行的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寿命信号蛋白，降解了错误折叠和受损的蛋白质（其积累对细胞有毒），并分解了外源蛋白质以产生抗原肽呈递给免疫系统。它是理解许多人类疾病，特别是癌症和神经退行性疾病，例如亨廷顿病的机制的基础。真核生物26 S蛋白酶体是由一个20 S蛋白酶体和两个19 S调节颗粒组成的，每个19 S调节颗粒含有6个与20 S蛋白酶体接触的ATP酶。ATP酶的一个关键作用是打开20 S中的门控通道以促进底物进入进行破坏。由于19 S调节颗粒的大尺寸和动态性质，尽管做出了大量努力，但用于结构测定的整个26 S蛋白酶体的结晶仍然不成功，并且ATP酶控制20 S中的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ATP酶-20 S复合物的结晶。在与哈佛医学院的Alfred Goldberg教授的合作中，我们发现ATP酶只需要它们的C-末端来诱导门打开。因此，我们将ATP酶诱导门开放的机制研究与ATP酶的结构测定分开。本论文主要研究了蛋白酶体ATP酶的两个关键问题：（1）ATP酶如何在20 S中打开门;（2）ATP酶在ATP酶循环过程中的构象变化。我们的目标是明确的，我们的方法是新颖的，独特的，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-末端诱导古细菌20 S的构象变化，导致其大门打开。在目标1中，我们将探讨古细菌20 S中支配这种构象变化的决定因素。在目标2中，我们将确定是否真核生物19 S ATP酶的C-末端触发类似的构象变化，导致在真核生物20 S的门打开。在目的3中，我们将试图阐明全长蛋白酶体ATP酶在其ATP酶循环过程中的构象变化。这些目标的实质性完成将推进我们对蛋白酶体介导的蛋白质降解的认识，蛋白酶体介导的蛋白质降解在许多人类疾病的发病机制中起着关键作用。它还将推进单粒子cryoEM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分子机器降解。蛋白质降解过程受到严格调控，并且在许多人类疾病，特别是癌症和神经退行性疾病，例如亨廷顿病的发病机制中起关键作用。本申请研究了蛋白酶体ATP酶调节蛋白酶体的蛋白水解活性的机制。

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.