SUMO-dependent Regulation of Ubiquitin Ligases in Genomic Stability

描述(申请人提供)：泛素和小泛素样修饰物(SUMO)，在基因组稳定性和对环境毒素的反应中具有关键但经典的独特作用。泛素和相扑通过E3连接酶共价连接到靶蛋白上，动态调节细胞的蛋白质组。泛素和相扑途径功能障碍导致人类遗传不稳定和癌症易感性。我们最近发现了相扑靶向的E3泛素连接酶家族(STUbls)，它泛素化并靶向蛋白酶体进行降解。这种意想不到的现象需要对相扑函数的频谱进行修改。这项修订后的提案建立在我们发现的基础上，并增加了直接将STUbL机制与癌症和基因组稳定性联系起来的新数据。我们已经确定了裂解酵母STUbL突变体基因组不稳定性的主要介体，并将通过这些靶点来表征STUbL的细胞和分子机制。来自裂解酵母的结果将为对人类和小鼠STUbL靶点进行研究提供一个框架。值得注意的是，这些靶点将STUbL的作用机制置于细胞生长/凋亡途径、基因组稳定性和肿瘤发生的交叉点。我们的两个具体目标集中在确定STUbL功能的分子机制上，包括在体外和体内。在目标1中，我们将通过控制关键相扑结合物的命运来检验STUbL作为细胞凋亡与DNA修复途径的守门人的假设。在目标2中，我们将解决我们的总体假设，即STUbL是细胞主键，通过特定和可定义的蛋白质相互作用界面控制基因组的稳定性。为了实现我们的目标，我们将把强大的酵母遗传学与尖端结构分析、MudPIT质谱学、生物化学和哺乳动物细胞生物学相结合。预期的结果将为包括RNF4在内的STUbL的基因组稳定作用提供关键的机械性见解，并可能为RNF4相关人类疾病的治疗策略提供建议。此外，对STUbL功能的详细了解应该会对基因毒性应激的反应产生普遍的影响。与公共卫生相关：我们已经发现了一个称为STUbL的关键细胞蛋白家族，它可以维持遗传稳定并调节癌症治疗反应。我们将揭示STUbL可能如何抑制肿瘤发生，从而扩大我们对细胞生长控制的知识，并在控制失败时指导治疗策略。

DESCRIPTION (provided by applicant): Ubiquitin and the small ubiquitin-like modifier (SUMO), have critical but classically distinct roles in genome stability and responses to environmental toxins. Ubiquitin and SUMO are covalently attached by E3 ligases to target proteins, to regulate the cell's proteome dynamically. Ubiquitin and SUMO pathway dysfunction results in genetic instability and cancer predispositions in humans. We recently discovered the SUMO-targeted E3 ubiquitin ligase family (STUbLs), which ubiquitinate and target sumoylated proteins to the proteasome for degradation. This unanticipated phenomenon requires a revision to the spectrum of SUMO functions. This revised proposal builds upon our discovery and adds new data that directly connects the STUbL mechanism to cancer and genomic stability. We have identified the main mediators of genomic instability in fission yeast STUbL mutants and we will characterize the cellular and molecular mechanisms of STUbLs through these targets. Results from fission yeast will provide a framework for studies conducted on the human and mouse STUbL targets. Notably, these targets place the STUbL mechanism of action at the intersection of cell growth/apoptotic pathways, genomic stability and oncogenesis. Our two Specific Aims focus on defining the molecular mechanisms of STUbL function both in vitro and in vivo. In Aim 1, we will test the hypothesis that STUbLs act as gatekeepers for apoptosis versus DNA repair pathways, by controlling the fates of key SUMO conjugates. In Aim 2, we will address our overall hypothesis that STUbLs are cellular master keys, controlling genome stability through specific and definable protein interaction interfaces. To attain our Aims we will integrate powerful yeast genetics with cutting-edge structural analyses, MudPIT mass spectrometry, biochemistry and mammalian cell biology. The expected results will provide critical mechanistic insights into the genome stabilizing roles of STUbLs including RNF4, and may suggest therapeutic strategies for RNF4- linked human diseases. Additionally, a detailed understanding of STUbL function should yield general implications for responses to genotoxic stress. PUBLIC HEALTH RELEVANCE: We have discovered a key family of cellular proteins called STUbLs that maintain genetic stability and mediate cancer therapeutic responses. We will uncover how STUbLs likely suppress tumorigenesis, thus expanding our knowledge of cell growth control and guiding therapeutic strategies when control fails.