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-蛋白酶体途径的ATP依赖性蛋白质降解去除在细胞过程调节中至关重要的短寿信号蛋白，降解错误折叠和受损的蛋白质，其积累对细胞有毒，并分解外来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的机制的基础，特别是癌症和神经退行性疾病，如亨廷顿病。真核26S蛋白酶体是由一个20S蛋白酶体组成的，其蛋白水解活性位点在其内部，两个19S调节颗粒各含有6个与20S接触的atp酶。atp酶的一个关键作用是在20S中打开门控通道，促进底物进入破坏。由于19S调节颗粒的大尺寸和动态性，尽管付出了巨大的努力，但整个26S蛋白酶体的结晶用于结构测定仍未成功，并且ATPases控制20S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：单粒子电子低温显微镜（cryoEM），它不需要蛋白酶体ATPases-20S复合物的结晶。通过与哈佛医学院的Alfred Goldberg教授的合作，我们发现atp酶只需要它们的c末端就能诱导门打开。因此，我们将atp酶诱导的门打开的机制研究与atp酶的结构测定分离开来。本应用主要关注蛋白酶体atp酶的两个关键问题：(1)atp酶如何在20S中打开门；(2)atp酶在atp酶周期中的构象变化。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，确定了ATPases的c端诱导古生菌20S的构象变化，导致其门打开。在目标1中，我们将探索控制古细菌20S中这种构象变化的决定因素。在Aim 2中，我们将确定真核生物19S atp酶的c端是否触发了类似的构象变化，从而导致真核生物20S中的门打开。在Aim 3中，我们将试图阐明全长蛋白酶体atp酶在其atp酶周期中的构象变化。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。它还将推动单粒子低温电子显微镜技术实现更高的分辨率，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一种叫做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，在许多人类疾病的发病机制中起关键作用，特别是癌症和神经退行性疾病，如亨廷顿病。本应用程序研究了蛋白酶体atp酶调节蛋白酶体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.