Mass Spectrometry-Based Approaches to Study Protein Ubiquitination

描述（由申请人提供）：泛素系统的复杂性需要先进的方法/方法来鉴定泛素化底物，揭示靶向机制的多样性，并阐明控制下游步骤的途径。从蛋白质组水平上的泛素-蛋白质结合物分析到单个多聚泛素链的定量分析，质谱技术正在泛素生物学领域取得重大进展。这些进展阐明了其他重要的未解决问题。例如，虽然已经鉴定了某些蛋白质的泛素修饰位点，但对于大多数泛素底物，赖氨酸受体位点尚不清楚。这些网站的意识提供了具体的证据修改和有针对性的研究的机会。许多研究小组已经观察到，通常情况下，同一底物内的多个赖氨酸被修饰。由于一个单一的多聚泛素链代表一个主管降解信号，额外的复杂性的目的是不清楚的。这种调控复杂性的一部分可能归因于几种主要的蛋白酶体相关泛素受体的作用，包括Dsk 2，Rad 23和Ddi 1，通常认为它们调节底物识别。为了揭示这些受体蛋白的底物特异性调节机制，需要鉴定其降解依赖于每个受体蛋白的缀合物的子集。该提案详细说明了我们将如何开发一种新的策略来确定泛素化位点。此外，我们建议扩展我们目前的方法，并精确地量化的泛素化的量发生在个别基板赖氨酸，使假设驱动的实验在体内和体外的基板特异性的方式。最后，我们将结合联合收割机稳定同位素标记与鸟枪蛋白质组学，以确定和区分底物的酵母泛素受体蛋白（UBL/UBA含），包括Rad 23，Dsk 2，和Ddi 1，为类似的努力在哺乳动物细胞。总之，这些整合的蛋白质组学策略将提供新的见解有关的作用，泛素受体蛋白和它们的影响，多聚泛素链的合成，结构和功能的生理相关的泛素底物。公共卫生相关性通过泛素连接对蛋白质进行修饰，产生一种信号，用于细胞的各种目的。泛素结合蛋白的研究最多的结果是蛋白质的降解（蛋白水解）。然而，非蛋白水解事件也是激烈的研究工作的主题。泛素系统研究的几乎每一个方面都显示出改善人类健康的巨大希望。除了其在正常生理学中的关键作用外，蛋白质泛素化的功能障碍还被认为是导致许多疾病和综合征（如癌症和阿尔茨海默病）的一个促成因素。该提案将提供新的技术来测量泛素生物学中的关键终点，包括多聚泛素（多泛素化）链形成的复杂结构和功能以及发现泛素化蛋白受体的底物。

DESCRIPTION (provided by applicant): The complexity of the ubiquitin system requires advanced approaches/methodologies to identify ubiquitinated substrates, uncover diversity in targeting mechanisms, and elucidate pathways that control downstream steps. From profiling ubiquitin-protein conjugates levels on a proteome scale to the quantitative analysis of individual polyubiquitin chains, mass spectrometry is making critical advances in ubiquitin biology. These advances illuminate other important unsolved issues. For example, although ubiquitin modification sites have been identified for some proteins, for most ubiquitin substrates, lysine-acceptor sites are not yet known. Awareness of these sites provides both concrete evidence of modification and the opportunity for targeted studies. Many groups have observed that more often than not, multiple lysines within the same substrate are modified. Since a single polyubiquitin chain represents a competent degradation signal, the purpose of the additional complexity is unclear. Part of this regulatory complexity is likely attributable to the roles of several prominent proteasome-associated ubiquitin receptors, including Dsk2, Rad23, and Ddi1, commonly believed to modulated substrate recognition. To uncover the mechanism of substrate-specific regulation by these receptor proteins, the subsets of conjugates whose degradation is dependent on each will need to be identified. This proposal details how we will develop a novel strategy to determine ubiquitination sites en masse. In addition, we propose to extend our current methods and precisely quantify the amount of ubiquitination occurring on individual substrate lysines enabling hypothesis-driven experiments in vivo and in vitro in a substrate-specific manner. Finally, we will combine stable isotope labeling with shotgun proteomics to identify and differentiate substrates for yeast ubiquitin receptor proteins (UBL/UBA-containing) including Rad23, Dsk2, and Ddi1, setting the stage for similar endeavors in mammalian cells. Together, these integrated proteomics strategies will provide new insights concerning the role of ubiquitin receptor proteins and their influence on polyubiquitin chain synthesis, structure, and function for physiologically relevant ubiquitin substrates. PUBLIC HEALTH RELEVANCE The modification of a protein by ubiquitin attachment produces a signal utilized with cells for a variety of purposes. The most studied outcome of a ubiquitin-conjugated protein is the degradation (proteolysis) of the protein. However, nonproteolytic eventualities are also the subject of intense research efforts. Nearly every aspect of research within the ubiquitin system shows great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in protein ubiquitination have been implicated as a contributing factor in the causation of many diseases and syndromes as diverse as cancer and Alzheimer's disease. This proposal will provide new technologies to measure critical end points in ubiquitin biology including the complex structure and function of polyubiquitin (multiple ubiquitination) chain formation and the discovery of substrates for ubiquitinated protein receptors.