Proteomics Approaches to Protein Turnover

 描述(申请人提供)：该项目的目标是开发蛋白质组学技术，用于全面测量细胞中的蛋白质周转，重点分析蛋白质丰度、泛素化、合成速率和降解率。根据分子生物学的中心教条，遗传信息从基因组到转录组再到蛋白质组。从理论上讲，细胞系统中的蛋白质动力学产生一种稳定状态，其中蛋白质的水平取决于其先前存在的浓度、合成速率和降解速率。真核细胞中的蛋白质降解主要由泛素(Ub)-蛋白酶体系统和自噬-溶酶体途径介导。泛素是一种由76个氨基酸组成的小蛋白质，可修饰数千种蛋白质，作为蛋白酶体降解和其他下游事件的多功能信号。泛素以单体和聚合物的形式结合到底物上。在这一应用中，我们建议开发基于MS的新方法来描述蛋白质周转，分析泛素化的蛋白质组和泛素链结构，并研究在病理生理条件下蛋白质周转是如何受到影响的。我们的四个具体目标是：(I)开发一种测量蛋白质组周转的20-plex集成MS方法；(Ii)开发分析多泛素链结构的中下MS方法；(Iii)研究不同的多泛素链在酵母和哺乳动物细胞中的功能；以及(Iv)研究人类疾病小鼠模型中蛋白质周转的变化。蛋白质周转和泛素化是基本的调节事件，有助于人类疾病的发病。这项研究将导致开发新的MS技术来研究蛋白质周转，并对多泛素链功能及其与人类疾病的关系有新的理解。

 DESCRIPTION (provided by applicant): The goal of this project is to develop proteomics technologies for the complete measurement of protein turnover in cells, focusing on the analysis of protein abundance, ubiquitination, synthetic rate, and degradation rate. According to the central dogma of molecular biology, genetic information flows from the genome to the transcriptome to the proteome. Theoretically, protein dynamics in a cellular system yields a steady state in which the level of a protein depends on its preexisting concentration, synthetic rate, and degradation rate. Protein degradation in eukaryotic cells is mainly mediated by the ubiquitin (Ub)-proteasome system and the autophagy-lysosome pathway. Ubiquitin, a small protein of 76 amino acids, modifies thousands of proteins as multifunctional signals for proteasomal degradation and other downstream events. Ubiquitin is conjugated to substrates in the form of monomers and polymers. In this application, we propose to develop novel mass spectrometry (MS)-based methods to profile protein turnover, analyze ubiquitinated proteome and ubiquitin chain structures, and study how protein turnover is affected under pathophysiological conditions. Our four specific aims are to (i) develop a 20-plex integrated MS approach for measuring proteome turnover; (ii) develop middle-down MS methods for analyzing polyubiquitin chain structures; (iii) study the function of diverse polyubiquitin chains in yeast ad mammalian cells; and (iv) investigate protein turnover alterations in mouse models of human disease. Protein turnover and ubiquitination are fundamental regulatory events, contributing to the pathogenesis of human disease. The research will lead to the development of novel MS technologies for studying protein turnover and new understanding of polyubiquitin chain function, as well as its involvement in human disease.