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/方法来识别泛素化底物，揭示靶向机制的多样性，并阐明控制下游步骤的途径。从在蛋白质组尺度上分析泛素蛋白偶联物水平到单个多泛素链的定量分析，质谱法在泛素生物学方面取得了重大进展。这些进展阐明了其他重要的未解决问题。例如，虽然已经确定了一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。对这些地点的认识既提供了修改的具体证据，也为有针对性的研究提供了机会。许多研究小组观察到，同一底物中的多个赖氨酸经常被修饰。由于单个多泛素链代表一个有效的降解信号，因此增加复杂性的目的尚不清楚。这种调控的复杂性部分可能归因于几种重要的蛋白酶体相关泛素受体的作用，包括Dsk2、Rad23和Ddi1，它们通常被认为调节底物识别。为了揭示这些受体蛋白对底物特异性调节的机制，需要鉴定其降解依赖于每个受体蛋白的偶联物亚群。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，并精确量化发生在单个底物赖氨酸上的泛素化量，从而以底物特异性的方式在体内和体外进行假设驱动的实验。最后，我们将结合稳定同位素标记和散弹枪蛋白质组学来鉴定和区分酵母泛素受体蛋白（含UBL/ uba）的底物，包括Rad23、Dsk2和Ddi1，为在哺乳动物细胞中进行类似的研究奠定基础。总之，这些整合的蛋白质组学策略将为泛素受体蛋白的作用及其对多泛素链合成、结构和生理相关泛素底物功能的影响提供新的见解。泛素附着对蛋白质的修饰产生一种信号，用于各种目的的细胞。泛素结合蛋白研究最多的结果是蛋白质的降解（蛋白质水解）。然而，非蛋白质水解的可能性也是激烈的研究努力的主题。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。除了其在正常生理中的关键作用外，蛋白质泛素化的功能障碍已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的关键终点，包括多泛素（多泛素化）链形成的复杂结构和功能以及泛素化蛋白受体底物的发现。

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.