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

翻译后摘要：在真核生物中，ATP依赖的蛋白质降解的泛素-蛋白酶体途径 去除在调节细胞过程中至关重要的短寿命信号蛋白，降解错误折叠， 受损蛋白质，其积累对细胞有毒，并分解外来蛋白质产生 用于呈递给免疫系统的抗原肽。它是理解这一机制的基础 许多人类疾病，特别是癌症和神经变性疾病，例如亨廷顿病。 真核生物26 S蛋白酶体是由具有蛋白水解活性位点的20 S蛋白酶体组成 两个19 S调节颗粒，每个颗粒含有与20 S接触的六个ATP酶。一 ATP酶的关键作用是打开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，整个26 S的结晶 蛋白酶体的结构确定仍然不成功，尽管大量的努力， 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 不需要蛋白酶体ATP酶-20 S结晶的电子冷冻显微镜（cryoEM） 复杂.在与来自哈佛医学院的阿尔弗雷德·戈德堡教授的合作中，我们发现， ATP酶仅需要它们的C-末端来诱导门打开。因此，我们将机械的 从ATP酶的结构测定研究ATP酶诱导的门开放。本申请 着重讨论了蛋白酶体ATP酶的两个关键问题：（1）ATP酶如何在20秒内打开门， (2)ATP酶在ATP酶循环过程中的构象变化。我们的目标明确，我们的 这种方法是新颖的，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中的构象变化，其导致 门的打开。 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 目的2：确定真核生物19 S ATP酶的C-末端是否引发类似的构象变化 导致真核细胞20 S中的门打开。在目标3中，我们将试图阐明构象 蛋白酶体ATP酶在ATP酶循环中的变化。基本实现这些目标 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蛋白质降解中起着关键作用。 许多人类疾病的发病机制。它还将推进单粒子cryoEM技术， 实现更高的分辨率和检测小的配体，只有几个残基的大小。

Abstract: 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.