MECHANISM OF CHAPERONE-ASSISTED ASSEMBLY OF PROTEASOME REGULATORY PARTICLE

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 蛋白酶体是主要的细胞蛋白酶。它参与蛋白质的受控降解，调节多种细胞过程，如转录、细胞凋亡、细胞分裂和 DNA 修复。由于在许多蛋白质的稳态中发挥着重要作用，因此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，这一点并不奇怪。细胞蛋白酶体活性的决定因素之一是细胞中蛋白酶体的水平。因此，了解细胞蛋白酶体水平如何调节以及蛋白酶体组装如何调节非常重要。该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种促进蛋白酶体调节颗粒（RP）形成的伴侣。每个伴侣与位于 RP 中的特定 AAA-ATP 酶的 C 结构域结合。尽管功能和结合特性相似，但这些伴侣之间不存在序列或结构保守性。 本文提出的研究目的是了解每个 RP 伴侣在蛋白酶体形成中所起的作用。我们假设伴侣分子充当 RP 组装中特定碱基亚基的模板。其次，它们调节前体复合物以空间和时间方式组装的顺序。我们还假设分子伴侣之间的结构差异对于在组装过程中适应和防止特定 ATP 酶的一组独特的相互作用非常重要。我们将使用体外结合和重构测定来研究这一点。此外，我们还将获得分子伴侣与其结合的 C 结构域的结构信息。我们期望这里提出的实验信息将提供对分子伴侣协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实现治疗手段。

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.