Regulation by Proteolysis-Independent Ubiquitination

描述（由申请人提供）：泛素化，即小蛋白泛素与其他蛋白质的共价连接，调节许多细胞过程。蛋白质泛素化 已成为蛋白质降解的同义词，目前的研究大多集中在泛素在靶向蛋白质以被26 S蛋白酶体降解中的作用。然而，我们开始意识到，许多蛋白质的泛素化的蛋白水解独立的方式进行调节。最近的系统范围的实验表明，只有略多于百分之五十的泛素化蛋白质被蛋白酶体有效地降解，这意味着蛋白质泛素化具有广泛的信号转导功能的蛋白酶体途径之外。这些蛋白水解无关的泛素信号分子的理解将是重要的基础生物医学研究和治疗靶向泛素系统的发展。一些关键问题是：为什么有些泛素化蛋白质被降解，而另一些则没有？泛素化如何直接影响蛋白质活性？通过泛素化直接调节蛋白质的机制是什么？调节的成分是什么？ 我们分析了由cullin-RING泛素连接酶复合物SCFMet 30调节的系统，该复合物将代谢应激与细胞周期调节联系起来。该途径特别适合于探测泛素化的非蛋白水解信号，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，然而一些底物被标记为降解，而其他底物以蛋白水解独立的方式被调节。该建议建立在可用于分析该途径的生物化学和生理学的大量工具的基础上，并且将解决（i）受调节的泛素结合结构域如何支配信号身份以及在泛素链信号传导之间切换以进行降解和非蛋白水解调节（Aim 1）;（ii）多聚泛素链如何直接调节转录因子活性（Aim 2）和（iii）泛素化如何诱导主动拆卸/重塑多亚基蛋白质复合物以调节其活性（目的3）。 泛素化影响许多重要的细胞过程，并与许多人类疾病，包括癌症，神经变性和逆转录病毒感染有关。这些疾病中的蛋白水解独立的泛素化的贡献正在出现，这将是重要的是要了解这种调节背后的机制，以设计诊断工具和治疗策略。该提案旨在实现详细的机制洞察蛋白水解独立的泛素信号，并定义这些监管泛素化途径的概念。 公共卫生相关性：泛素修饰的蛋白质控制着活细胞的大多数功能，并参与许多人类疾病。该项目旨在从机制上理解蛋白质功能通过泛素化的调节和泛素化机制的关键组分的调节。泛素系统是一个很有前途的治疗方法的目标，因此，从拟议的研究中得出的结果将对人类健康具有重要意义。

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. PUBLIC HEALTH RELEVANCE: Protein modification with ubiquitin controls most if not all functions of living cells and is involved in many human diseases. This project seeks a mechanistic understanding of regulation of protein function by ubiquitylation and regulation of key components of the ubiquitylation machinery. The ubiquitin system is a promising target for therapeutic approaches and findings derived from the proposed studies will therefore be of great significance to human health.