Proteomics of the proteasome interacting networks

描述（由申请人提供）：当前蛋白质组学研究的主要目标之一是通过全局绘制蛋白质-蛋白质相互作用来阐明蛋白质功能。全面了解大分子蛋白质复合物功能的关键一步是表征蛋白质复合物的组成并绘制其相互作用网络。基于质谱的相互作用蛋白质组学已成为分析功能蛋白质复合物的首选方法。为了捕获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质谱的蛋白质组学方法来破译蛋白质相互作用网络的动力学。26 S蛋白酶体是一种大分子机器，负责细胞质和细胞核中的泛素/ATP依赖性蛋白质降解。泛素-蛋白酶体介导的蛋白质降解在调节许多生物学过程中是必不可少的，包括细胞分裂、转录、细胞信号传导和发育。正常的泛素-蛋白酶体降解途径的破坏与广泛的人类疾病有关。尽管有大量的研究，许多关键问题仍然没有答案，特别是泛素化底物如何被26 S蛋白酶体识别和转运的机制。为了解决这些问题，我们假设存在多种泛素受体或替代途径，并负责调节底物识别和运输到26 S蛋白酶体进行降解。 为了验证这一假设，我们打算应用一种新的集成质谱为基础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，并确定蛋白酶体及其底物之间的分子连接。具体目标是：1）开发和优化一种新的整合蛋白质组学方法，利用体内交联、亲和纯化和质谱分析来捕获和鉴定动态蛋白酶体相互作用网络; 2）鉴定与细胞周期不同阶段相关的特异性蛋白酶体相互作用蛋白，以及在检查点诱导的细胞周期停滞中; 3）通过蛋白酶体亚基与其相互作用伴侣的交叉定位，连接的肽序列，并产生蛋白酶体复合物的空间组织和与其相互作用伴侣的相互作用连接，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.