Quantitative mapping of ubiquitin ligase substrates

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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.