Quantitative mapping of ubiquitin ligase substrates

泛素化是一种重要的翻译后修饰，它调节了大量的生物过程，包括蛋白质降解、信号转导、蛋白质运输和DNA修复。泛素（Ub）在底物蛋白上的共价附着是由Ub连接酶催化的，这种连接酶有数百种之多。细胞生物学中一个紧迫的问题是识别泛素化目标和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在一定程度上解决了这个问题。然而，目前还没有一种简单明了的方法来匹配特定的Ub连接酶与特定的目标底物。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：通过采用新设计的定量无标记质谱技术，开发一种简化的策略来创建酶显性负E3连接酶，并跟踪它们诱导的蛋白质组和“泛素组”的变化。泛素化是一种重要的翻译后修饰，它调节了大量的生物过程，包括蛋白质降解、信号转导、蛋白质运输和DNA修复。一个巨大的进步将是知道每个特定的泛素连接酶修饰的蛋白质。我们将开发一种新技术，通过跟踪一组新的“逆转”Ub连接酶如何影响酵母细胞中泛素化蛋白的水平来实现这一目标。这将建立一个总体策略，绘制出哪些连接酶靶向哪些底物，并有助于揭示泛素在生物学中扮演的多重角色。

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.