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。蛋白酶体生物学中的一个重要问题是如何有效地从CP中释放蛋白水解产物的短肽，以确保大蛋白底物降解所需的连续底物进入和产物释放。一个被广泛接受但未经测试的范例是26 S蛋白酶体单向发挥功能，其中未折叠底物从一端进入CP，蛋白水解产物从相对端离开。另一个重要的问题是，在Rpt环组装过程中，Rpt亚基对ATP的水解作用是什么，以及组装是否需要CP作为模板？在本申请中，我们旨在解决这些问题。 我们将使用近原子分辨率的单粒子cryoEM作为我们的主要结构分析工具，结合分子生物学，生物化学和生物物理工具中的其他方法，来阐明调节对称蛋白质降解机制的不对称功能的机制。具体目的是（1）确定蛋白水解产物从20 S降解室释放的机制，（2）确定与20 S核心颗粒相对末端结合的蛋白酶体激活剂的功能协调机制，（3）确定ATP水解在真核蛋白酶体ATP酶组装途径中的作用。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。

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. An important question in proteasome biology is that how short peptides of proteolytic products are released efficiently from CP to ensure a continuous substrate entering and products release required for the degradation of large protein substrates. A widely accepted yet untested paradigm is that the 26S proteasome functions unidirectional in which unfolded substrates enter the CP from one end and the proteolytic products exit from the opposite end. Another important question is what is the role of ATP hydrolysis by Rpt subunits during the Rpt ring assembly, and if the assembly requires CP as a template? In this application, we aim to address these questions. We will use near atomic resolution single particle cryoEM as our main structural analysis tool, together with other methods in molecular biology, biochemistry and biophysical tool, to elucidate the mechanisms that regulates the asymmetrical functionality of the symmetrical protein degradation machinery. The specific aims are (1) determine the mechanism of proteolytic products releasing from the 20S degradation chamber, (2) determine mechanism that coordinates the functions of proteasomal activators bound to the opposite ends of 20S core particle, and (3) determine the role of ATP hydrolysis in the assembly pathway of eukaryotic proteasomal ATPases. 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.