Regulation by Proteolysis-Independent Ubiquitination

描述(申请人提供)：泛素化，小蛋白泛素与其他蛋白质的共价连接，调节一系列细胞过程。蛋白质泛素化 已经成为蛋白质降解的同义词，目前的研究大多集中在泛素在26S蛋白酶体靶向降解蛋白质中的作用。然而，我们开始认识到，一些蛋白质是由泛素化以一种不依赖于蛋白质降解的方式调节的。最近的全系统实验表明，只有略多于50%的泛素化蛋白质被蛋白酶体有效降解，这意味着蛋白质泛素化在蛋白酶体途径之外具有广泛的信号功能。对这些不依赖于蛋白质降解的泛素信号的分子理解将对基础生物医学研究和针对泛素系统的治疗药物的开发具有重要意义。其中一些关键问题是：为什么一些泛素化的蛋白质会被降解，而另一些不会？泛素化是如何直接影响蛋白质活性的？泛素化直接调节蛋白质的机制是什么？调节的成分是什么？我们分析了一个由剔除环泛素连接酶复合体SCFMet30调控的系统，该复合体将代谢应激与细胞周期调节联系起来。这种途径特别适合于探测泛素化的非蛋白水解性信号，因为相同的连接酶用相同的赖氨酸-48连接的泛素链修饰不同的底物蛋白质，而一些底物被标记为降解，而另一些底物以不依赖于蛋白降解的方式调节。这一建议建立在可用于分析这一途径的生化和生理学的大量工具的基础上，并将涉及(I)受调控的泛素结合域如何决定信号识别以及泛素链如何在发出降解信号的泛素链和非蛋白水解性调控之间切换(目标1)；(Ii)多泛素链如何直接调节转录因子的活性(目标2)；以及(Iii)泛素化如何诱导多亚单位蛋白质复合体的主动拆解/重塑以调节其活性(目标3)。泛素化影响许多重要的细胞过程，并与许多人类疾病有关，包括癌症、神经退行性变和逆转录病毒感染。蛋白降解非依赖泛素化在这些疾病中的作用正在显现，了解这一调节背后的机制以设计诊断工具和治疗策略将是重要的。这项建议旨在实现对蛋白质降解非依赖泛素信号的详细机制洞察，并定义这些调节泛素化途径的概念。

DESCRIPTION (provided by applicant): Ubiquitylation, the covalent attachment of the small protein ubiquitin to other proteins, regulates a host of cellular processes. Protein ubiquitylation has become a synonym for protein degradation, and most of the current research is focused on the role of ubiquitin in targeting proteins for degradation by the 26S proteasome. However, we are beginning to appreciate that a number of proteins are regulated by ubiquitylation in a proteolysis-independent manner. Recent system-wide experiments suggest that only little more than fifty percent of ubiquitylated proteins are efficiently degraded by the proteasome, implying that protein ubiquitylation has widespread signaling functions outside the proteasome pathway. Molecular understanding of these proteolysis-independent ubiquitin signals will be important for basic biomedical research and development of therapeutics targeting the ubiquitin system. Some of the key questions are: Why are some ubiquitylated proteins degraded and others are not? How can ubiquitylation directly affect protein activity? What are the mechanisms of direct protein regulation by ubiquitylation and what are the components mediating regulation? We analyze a system regulated by the cullin-RING ubiquitin ligase complex SCFMet30, which connects metabolic stress to cell cycle regulation. This pathway is particularly suited to probe non-proteolytic signals of ubiquitylation because the same ligase modifies different substrate proteins with the same lysine-48 linked ubiquitin chain, yet some substrates are labeled for degradation while other substrates are regulated in a proteolysis-independent manner. This proposal builds on a plethora of tools available to analyze biochemistry and physiology of this pathway and will address (i) how regulated ubiquitin-binding domains can dictate signal identity and switch between ubiquitin chains signaling for degradation and non-proteolytic regulation (Aim 1); (ii) how a polyubiquitin chain can directly regulate transcription factor activity (Aim 2) and (iii) how ubiquitylation induces active disassembly/remodeling of multisubunit protein complexes to modulate their activities (Aim 3). Ubiquitylation affects many important cellular processes and has been linked to a number of human diseases including cancer, neurodegeneration, and retroviral infection. A contribution of proteolysis-independent ubiquitylation in these diseases is emerging and it will be important to understand the mechanism behind this regulation to design diagnostic tools and treatment strategies. This proposal aims to achieve detailed mechanistic insight into proteolysis-independent ubiquitin signals and to define the concepts of these regulatory ubiquitylation pathways.