Proteomics of the proteasome interacting networks

描述(申请人提供)：当前蛋白质组学研究的主要目标之一是通过绘制蛋白质-蛋白质相互作用的全球图谱来阐明蛋白质的功能。要全面了解大分子蛋白质复合体的功能，关键的一步是表征蛋白质复合体的组成并绘制其相互作用网络图。基于质谱学的相互作用蛋白质组学已经成为分析功能蛋白质复合体的首选方法。为了捕捉在完整细胞中发生的所有蛋白质-蛋白质相互作用，我们将开发一种新的基于集成质谱学的蛋白质组学方法来破译蛋白质相互作用网络的动力学。26S蛋白酶体是一种大分子机器，负责胞浆和胞核中泛素/ATP依赖的蛋白质的降解。泛素-蛋白酶体介导的蛋白质降解在调节细胞分裂、转录、细胞信号和发育等许多生物学过程中起着至关重要的作用。正常泛素-蛋白酶体降解途径的中断已被认为与多种人类疾病有关。尽管进行了大量的研究，但仍有许多关键问题没有得到解答，特别是泛素化底物如何被26S蛋白酶体识别和转运到26S蛋白酶体的机制。为了解决这些问题，我们假设存在多个泛素受体或替代途径，负责调节26S蛋白酶体的底物识别和运输到26S蛋白酶体降解。为了验证这一假设，我们打算应用一种新的基于集成质谱学的蛋白质组学方法来破译蛋白质组范围内的蛋白酶体相互作用网络，并确定蛋白酶体与其底物之间的分子联系。其具体目标是：1)建立和优化一种新的集成蛋白质组学方法，通过体内交联、亲和纯化和质谱分析来捕获和鉴定动态的蛋白酶体相互作用网络；2)鉴定与细胞周期不同阶段和检查点诱导的细胞周期停滞相关的特定蛋白酶体相互作用蛋白；3)通过绘制交联肽序列图来确定蛋白酶体亚单位与其相互作用伙伴之间的蛋白质相互作用界面，并建立蛋白酶体复合体的空间组织以及与其相互作用伙伴的相互作用链接，以充分阐明它们的功能。

DESCRIPTION (provided by applicant): One of the major goals in current proteomics research is to elucidate protein functions by globally mapping protein-protein interactions. A key step toward gaining a full understanding of the function of a macromolecular protein complex is to characterize protein complex composition and map their interaction networks. Mass spectrometry-based interaction proteomics has become the method of choice for analyzing functional protein complexes. To capture all protein-protein interactions occurring in intact cells, we will develop a novel integrated mass spectrometry-based proteomics approach to decipher the dynamics of protein interaction networks. The 26S proteasome is a macromolecular machine responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. Ubiquitin-proteasome-mediated protein degradation is essential in regulating many biological processes including cell division, transcription, cell signaling and development. Disruption of normal ubiquitin-proteasome degradation pathways has been implicated in a wide range of human disease. Despite intensive research, many key questions remain unanswered, especially the mechanisms of how ubiquitinated substrates are recognized by and translocated to 26S proteasome. To address these questions, we hypotheses that multiple ubiquitin receptors or alternative pathways are present and responsible for regulating substrate recognition by and transport to the 26S proteasome for degradation. To test the hypothesis, we intend to apply a novel integrated mass spectrometry-based proteomics approach to decipher proteome-wide proteasome interacting networks and determine the molecular linkage between the proteasome and its substrates. The specific aims are: 1) to develop and optimize a novel integrated proteomics approach to capture and identify the dynamic proteasome interacting networks using in vivo cross-linking, affinity purification and mass spectrometry analysis; 2) to identify specific proteasome interacting proteins related to different phases of cell cycle and in a checkpoint-induced cell cycle arrest; 3) to identify the protein interaction interface between proteasome subunits and their interacting partners by mapping cross-linked peptide sequences and generate the spatial organization of the proteasome complexes and interaction linkage with their interacting partners to fully elucidate their functions.