Proteomics of the Proteasome Interacting Networks

描述（由申请人提供）：细胞中的许多过程由大蛋白质组装体或多蛋白质复合物介导，这些蛋白质组装体或多蛋白质复合物以高度调节的方式一起工作以维持正常的稳态。多亚基蛋白质复合物中的畸变可导致各种疾病状态。为了了解“蛋白质机器”在各种细胞过程中的功能，并帮助未来的药物开发，绘制蛋白质复合物的相互作用网络已成为现代蛋白质组学研究的主要努力之一。为了捕获活细胞中所有类型的蛋白质相互作用，我们开发了QTAX方法，以允许有效捕获、纯化和定量鉴定蛋白质复合物的稳定、弱和/或瞬时蛋白质相互作用。这种策略允许生成蛋白质相互作用网络的真实快照，因为它们存在于活细胞中。泛素-蛋白酶体系统（UPS）是真核生物细胞内蛋白质降解的主要调控途径，参与调控许多重要的生理过程。已知UPS的畸变会导致包括癌症在内的多种人类疾病。第一类抗癌药物通过对UPS的一般抑制起作用，并且是有效的，但不能用于长期治疗。这是因为一般的蛋白酶体抑制将阻断整个降解过程，并非特异性地影响许多过程。为了开发更有效和毒性更小的治疗方法，有必要确定蛋白酶体结构和功能的异质性，并获得更好地了解UPS调节和底物特异性的分子机制。为了实现这些目标，该提案的目的是研究两个以前未表征的亚组的人26 S蛋白酶体复合物在哺乳动物细胞和推进QTAX为基础的蛋白质相互作用的研究，以新的水平，通过定义在体内蛋白质复合物的结构拓扑结构。这些新策略的发展将是蛋白质组学研究中令人兴奋的技术进步。具体目标包括：1）使用QTAX策略阐明DNA结合和非DNA结合的人蛋白酶体复合物的结构和功能差异; 2）开发下一代QTAX策略，用于蛋白质复合物体内蛋白质相互作用拓扑结构的质谱表征; 3）通过分离标签策略和定量质谱法确定遍在蛋白受体相关蛋白酶体复合物的定量差异。

DESCRIPTION (provided by applicant): Many processes in cells are mediated by large protein assemblies or multi-protein complexes that work together in a highly regulated manner to maintain normal homeostasis. Aberrations in multi-subunit protein complexes can lead to various disease states. In order to understand how "protein machines" function in various cellular processes and to aid future drug development, mapping interaction networks of protein complexes has become one of the major endeavors in modern proteomics research. In order to capture protein interactions of all types in living cells, we have developed the QTAX method to allow effective capture, purification, and quantitative identification of stable, weak and/or transient protein interactions of protein complexes. This strategy allows generating an authentic snapshot of protein interaction networks as they exist in living cells. The ubiquitin-proteasome system (UPS) represents the major pathway for regulated degradation of intracellular proteins in eukaryotes, which helps control numerous essential physiological processes. Aberration of the UPS is known to lead to a variety of human diseases including cancer. The first class of anticancer drugs acts through general inhibition of the UPS and is effective but cannot be used for long-term treatment. This is because general proteasome inhibition will block the entire degradation process and affect many processes nonspecifically. In order to develop more effective and less toxic therapeutics, it is necessary to determine proteasome structural and functional heterogeneity and obtain a better understanding of the molecular mechanisms of UPS regulation and substrate specificity. Toward these goals, this proposal aims to investigate two previously uncharacterized subgroups of human 26S proteasome complexes in mammalian cells and to advance QTAX-based protein interaction study to new levels by defining structural topologies of in vivo protein complexes. The development of these novel strategies will be an exciting technological advancement in proteomics research. The specific aims include: 1) To unravel structural and functional differences of DNA-bound and non-DNA bound human proteasome complexes using the QTAX strategy; 2) To develop the next generation of the QTAX strategy for mass spectrometric characterization of in vivo protein interaction topologies of protein complexes; 3) To determine quantitative differences in ubiquitin receptor- associated proteasome complexes by a split-tag strategy and quantitative mass spectrometry.