Discovery of ubiquitin ligase substrates - the development of integrated experime

描述（由申请人提供）：泛素是细胞中最广泛使用的蛋白质共价修饰剂之一。蛋白质泛素化在细胞和发育生物学的几乎每个方面都起着关键的调节作用，我们已经认识到，特定的泛素化错误事件是许多使人衰弱和致命的人类疾病（包括癌症和神经退行性疾病）的基础。泛素化通过酶级联反应发生，其中泛素从泛素活化酶传递到泛素缀合酶，然后通过泛素连接酶连接到底物，这赋予底物特异性。因为我们对大多数泛素连接酶的底物群知之甚少，所以预测的近1000种人类泛素连接酶的功能还没有被表征。目前还没有常规技术来鉴定遍在蛋白连接酶的底物。这阻碍了泛素化领域以及其中泛素化起关键作用的领域的进展，例如干细胞生物学、癌症和神经退行性疾病。我们建议通过开发基于质谱的泛素蛋白质组学方法来鉴定泛素连接酶的底物，以解决这个总体的细胞生物学问题。为了发展我们的方法，我们将利用酵母（一种简单的真核生物）的遗传和生化易处理性。因为泛素化在所有真核生物中是保守的，所以大多数酵母泛素化途径都有人类对应物。因此，我们将学习有价值的生物信息，同时为开发这种技术的哺乳动物版本提供模板。我们的生物学目标的成功高度依赖于计算蛋白质组学。因此，我们建议泛素生物学家和计算科学家共同努力，以实现以下目标：（1）改进实验设计和发展统计方法，通过串联质谱（MS/MS）鉴定泛素连接酶的推定底物;（2）发展完善的实验方案，以减少泛素蛋白质组样品的复杂性;（3）扩展质谱数据的统计分析，以利用MS/MS和LC-MS数据的混合方法。 公共卫生相关性：泛素对细胞蛋白质的修饰是细胞严格控制蛋白质功能的最重要手段之一。蛋白质泛素化的应用非常广泛，人类细胞拥有近1000种泛素连接酶来对蛋白质进行这种关键修饰，但我们对这些酶中任何一种的底物群知之甚少。我们将开发尖端的实验和计算技术，以确定任何所需的泛素连接酶，特别是那些涉及人类疾病，如癌症和神经退行性疾病的底物队列。

DESCRIPTION (provided by applicant): Ubiquitin is one of the cell's most broadly deployed covalent modifiers of proteins. Protein ubiquitination serves critical regulatory roles in virtually every aspect of cell and developmental biology, and we've come to appreciate that particular ubiquitination misevents underlie many debilitating and deadly human diseases including cancer and neurodegenerative disorders. Ubiquitination occurs through an enzymatic cascade in which ubiquitin is passed from a ubiquitin activase to a ubiquitin conjugase, and then attached to a substrate by a ubiquitin ligase, which imparts the substrate specificity. Because we know little about the substrate cohort of most ubiquitin ligases, functions for the nearly 1000 predicted human ubiquitin ligases have not been characterized. No routine technologies currently exist to identify substrates of ubiquitin ligases. This hampers both the advancement of the ubiquitination field as well as fields in which ubiquitination serves a key role, such as stem cell biology, cancer, and neurodegenerative disorders. We propose to address this overarching cell biological problem by developing mass spectrometry- based ubiquitin proteomics methods to identify substrates for ubiquitin ligases. To develop our methodology, we will take advantage of the genetic and biochemical tractability of yeast, a simple eukaryote. Because ubiquitination is conserved in all eukaryotes, most yeast ubiquitination pathways have human counterparts. Consequently, we will learn valuable biological information while providing a template for the development of a mammalian version of this technology. The success of our biological objective is highly dependent on computational proteomics. We therefore propose a coordinated effort between ubiquitin biologists and computational scientists to accomplish the following aims: (1) Improve experimental design and develop statistical methods to identify putative substrates of ubiquitin ligases by tandem mass spectrometry (MS/MS); (2) Develop refined experimental protocols to reduce the complexity of the ubiquitin proteome samples; (3) Expand the statistical analysis of the mass spectrometry data to exploit a hybrid approach using MS/MS and LC-MS data. PUBLIC HEALTH RELEVANCE: The modification of cellular proteins by the ubiquitin is one of the cell's most important means to tightly control protein function. Protein ubiquitination is so broadly used that the human cell possesses nearly 1000 ubiquitin ligases to add this critical modification to proteins, but we know very little about the cohort of substrates for any one of these enzymes. We will develop cutting-edge experimental and computational technologies to identify the substrate cohorts for any desired ubiquitin ligase, especially those implicated in human diseases such as cancer and neurodegenerative disorders.