Proteomics of the proteasome interacting networks

蛋白酶体相互作用网络的蛋白质组学

基本信息

批准号：
7934387
负责人：
Lan Huang
金额：
$ 19.32万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7934387
关键词：
26S proteasome ATP phosphohydrolase Address Affinity Chromatography Apoptosis Binding Biological Biological Process Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Nucleus Cell division Cells Complex Consensus Cytosol DNA Repair Degradation Pathway Development Genetic Transcription Goals Malignant Neoplasms Mammalian Cell Maps Mass Spectrum Analysis Mediating Methodology Methods Molecular Multiprotein Complexes Pathway interactions Phase Physiological Protein Dynamics Protein Subunits Protein-Protein Interaction Map Proteins Proteome Proteomics Regulation Research Saccharomycetales Signal Transduction Site-Directed Mutagenesis Subgroup System Technology Testing UBA Domain Ubiquitin Ubiquitination base crosslink human disease in vivo link protein malignant neurologic neoplasms multicatalytic endopeptidase complex novel protein aminoacid sequence protein complex protein degradation protein function protein protein interaction receptor

项目摘要

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.

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球映射蛋白质 - 蛋白质相互作用来阐明蛋白质功能。全面了解大分子蛋白复合物的功能的关键步骤是表征蛋白质复合物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，我们将开发一种基于质谱质谱的新型蛋白质组学方法来解密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，负责细胞质和核中泛素/ATP依赖性蛋白质降解。泛素 - 蛋白酶体介导的蛋白质降解对于调节许多生物学过程至关重要，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。尽管进行了深入的研究，但许多关键问题仍未得到解决，尤其是如何通过泛素化底物识别并转移到26S蛋白酶体的机制。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，并负责通过26S蛋白酶体进行降解来调节底物识别和运输。为了检验假设，我们打算将基于质谱的新型蛋白质组学方法应用于解密的蛋白质组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链接。具体目的是：1）开发和优化一种新型的综合蛋白质组学方法，以使用体内交联，亲和力纯化和质谱分析来捕获和识别动态蛋白酶体相互作用网络； 2）确定与细胞周期不同阶段以及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的蛋白； 3）通过绘制交联的肽序列并生成蛋白酶体复合物的空间组织以及与其相互作用的伴侣的相互作用联系，以完全阐明其功能，以确定蛋白酶体亚基与其相互作用伙伴之间的蛋白质相互作用界面。