MECHANISM OF CHAPERONE-ASSISTED ASSEMBLY OF PROTEASOME REGULATORY PARTICLE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白酶体是细胞内主要的蛋白水解酶。它参与了蛋白质的受控降解，这些蛋白质调控着广泛的细胞过程，如转录、凋亡、细胞分裂和DNA修复。由于在许多蛋白质的动态平衡中起着重要作用，观察到蛋白酶体活性增加(例如在多发性骨髓瘤中)或蛋白酶体活性降低(例如在许多神经退行性疾病中)是许多疾病的病理因素也就不足为奇了。细胞蛋白酶体活性的一个决定因素是细胞中蛋白酶体的水平。因此，重要的是要了解细胞蛋白酶体水平是如何被调控的，以及蛋白酶体组装是如何被调控的。这个项目的长期目标是了解蛋白酶体组装的机制。最近的工作已经确定了四个促进蛋白酶体调节颗粒(RP)形成的伴侣。每个伴侣结合到位于RP中的特定AAA-ATPase的C-结构域。尽管在功能和结合性质上有这种相似性，但这些伴侣之间没有序列或结构上的保守性。 这项研究的目的是了解每个RP伴侣在蛋白酶体的形成中所起的作用。我们假设伴侣在RP组装中作为特定碱基亚单位的模板。其次，它们调节了前体复合体在空间和时间上聚集的顺序。我们还假设，在组装过程中，伴侣之间的结构差异对于适应和防止特定ATPase的一组独特的相互作用是重要的。我们将使用体外结合和重建试验来研究这一点。此外，我们还将获得伴侣的结构信息，并结合它们结合的C-结构域。我们期望从这里提出的实验中获得的信息将提供对伴侣用来辅助蛋白酶体组装的机制的详细了解。这可能会提供新的药物靶点，并最终使我们能够操纵蛋白酶体水平或活性，以用于治疗手段。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The proteasome is the major cellular protease. It is involved in the controlled degradation of proteins that regulate a wide variety of cellular processes, such as transcription, apoptosis, cell division and DNA repair. With an important role in homeostasis of so many proteins it is not surprising that observed increased proteasome activity (e.g. in multiple myelomas) or decreased proteasome activity (e.g. in many neurodegenerative diseases) is a pathological factor in many diseases. One determinant of cellular proteasome activity is the level of proteasomes in the cell. Thus, it is important to understand how cellular proteasome levels are regulated and how proteasome assembly is regulated. The long-term goal of this project is to understand the mechanisms of proteasome assembly. Recent work has identified four chaperones that facilitate the formation of proteasome regulatory particle (RP). Each chaperone binds to the C-domain of a specific AAA-ATPases located in the RP. Despite this similarity in function and binding properties, there is no sequence or structural conservation among these chaperones. The objective of the research proposed here is to understand the role each RP chaperone plays in the formation of proteasomes. We hypothesize that the chaperones act as templates for specific base subunits in the assembly of RP. Secondly, they regulate the order in which precursor complexes assemble in a spatial and temporal manner. We also hypothesize that the structural difference among the chaperones are important to accommodate as well as prevent a unique set of interactions for specific ATPases during the assembly process. We will use in vitro binding and reconstitution assays to study this. We furthermore will obtain structural information of the chaperones in combination with the C-domain they bind to. We expect that information from the experiments proposed here will provide a detailed understanding of the mechanisms the chaperones employ to assist proteasome assembly. This could potentially provide new drug targets and ultimately enable us to manipulate proteasome levels or activity for therapeutic means.