Proteomics of the Proteasome Interacting Networks

描述（由申请人提供）：细胞中的许多过程是由大蛋白组装或多蛋白复合物介导的，它们以高度调节的方式共同作用以维持正常的体内平衡。多亚基蛋白复合物的畸变可导致各种疾病状态。为了了解“蛋白质机器”如何在各种细胞过程中发挥作用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，可以有效地捕获，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。这种策略可以生成活细胞中存在的蛋白质相互作用网络的真实快照。泛素-蛋白酶体系统（UPS）是真核生物胞内蛋白降解调控的主要途径，有助于控制许多重要的生理过程。UPS的异常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，有效但不能用于长期治疗。这是因为一般的蛋白酶体抑制会阻断整个降解过程，并非特异性地影响许多过程。为了开发更有效、毒性更小的治疗方法，有必要确定蛋白酶体的结构和功能异质性，并更好地了解UPS调节和底物特异性的分子机制。为了实现这些目标，本研究旨在研究哺乳动物细胞中人类26S蛋白酶体复合物的两个先前未被表征的亚群，并通过定义体内蛋白复合物的结构拓扑将基于qtaxs的蛋白相互作用研究提升到新的水平。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)利用QTAX策略揭示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)通过分裂标签策略和定量质谱法确定泛素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.