Quantitative mapping of ubiquitin ligase substrates

泛素化是一种重要的翻译后修饰，它调节着蛋白质降解、信号转导、蛋白质运输和DNA修复等一系列生物学过程。泛素（Ub）与底物蛋白的共价连接是由Ub连接酶催化的，其数量可达数百种。细胞生物学中的一个紧迫问题是鉴定泛素化的靶标并确定负责特定泛素化事件的Ub连接酶。最近开发的几种蛋白质组学技术部分解决了这个问题。然而，尚未开发出将特定的Ub连接酶与特定的一组靶底物匹配的简单而明确的方法。本提案的目标是开发这样一种方法，并绘制出由Ub连接酶控制的调控网络。这项工作将包括以下目标：开发一种简化的策略，以创建酶显性负E3连接酶，并遵循蛋白质组和“泛素化”的变化，他们通过适应新设计的定量无标记质谱技术诱导。泛素化是一种重要的翻译后修饰，它调节着蛋白质降解、信号转导、蛋白质运输和DNA修复等一系列生物学过程。一个巨大的进步将是知道每种特定的泛素连接酶修饰哪些蛋白质。我们将开发一种新的技术来实现这一目标，通过以下一组新的“逆转”Ub连接酶如何影响酵母细胞中泛素化蛋白质的水平。这将建立一个通用的策略，以确定哪些连接酶靶向哪些底物，并帮助解开泛素在生物学中的多重作用。 公共卫生相关性：泛素化是一种重要的翻译后修饰，它调节着蛋白质降解、信号转导、蛋白质运输和DNA修复等一系列生物学过程。一个巨大的进步将是知道每种特定的泛素连接酶修饰哪些蛋白质。我们将开发一种新的技术来实现这一目标，通过以下一组新的“逆转”Ub连接酶如何影响酵母细胞中泛素化蛋白质的水平。这将建立一个通用的策略，以确定哪些连接酶靶向哪些底物，并帮助解开泛素在生物学中的多重作用。

Ubiquitination is an important post-translational modification that regulates a vast array of biological processes including protein degradation, signal transduction, protein trafficking and DNA repair. The covalent attachment of ubiquitin (Ub) onto substrate proteins is catalyzed by Ub ligases, which number well into the hundreds. One of the pressing questions in cell biology is the identification of ubiquitinated targets and determining the Ub ligase responsible for a particular ubiquitination event. Several proteomic techniques have been recently developed that partly address this question. However, a simple and clear method to match a particular Ub ligase with a particular set of target substrates has not yet been developed. The goal of this proposal is to develop such a method and to map out the regulatory network controlled by Ub ligases. This work will be encompassed by the following Aim: Develop a streamlined strategy to create enzymatically dominant-negative E3 ligases and follow changes in the proteome and "ubiquitome" they induce by adapting newly devised quantitative label-free mass-spectroscopy techniques. Ubiquitination is an important post-translational modification that regulates a vast array of biological processes including protein degradation, signal transduction, protein trafficking and DNA repair. A tremendous advance would be knowing what proteins each particular ubiquitin ligase modifies. We will develop a new technology to accomplish this by following how a novel set of "reversal" Ub ligases impact on the repertoire of levels ubiquitinated proteins in yeast cells. This will establish out a general strategy to map out which ligases target what substrates and help unravel the multiple roles ubiquitin plays in biology. PUBLIC HEALTH RELEVANCE: Ubiquitination is an important post-translational modification that regulates a vast array of biological processes including protein degradation, signal transduction, protein trafficking and DNA repair. A tremendous advance would be knowing what proteins each particular ubiquitin ligase modifies. We will develop a new technology to accomplish this by following how a novel set of "reversal" Ub ligases impact on the repertoire of levels ubiquitinated proteins in yeast cells. This will establish out a general strategy to map out which ligases target what substrates and help unravel the multiple roles ubiquitin plays in biology.