Regulation by Proteolysis-Independent Ubiquitination

说明(由申请人提供的用小蛋白泛素修饰蛋白质的过程，称为泛素化的过程，在大多数细胞过程中发挥着关键作用。泛素连接酶是泛素化机制中最复杂、最重要的组成部分。它们赋予底物选择性，是监管的主要目标。泛素连接酶(E3酶)的重要性被作为E3发挥作用的大量蛋白质所强调。据估计，人类基因组编码600-1000个具有泛素连接酶活性的蛋白质，这个数字明显高于蛋白激酶。泛素连接酶有望在代谢途径和其他细胞过程之间形成联系，如细胞周期、应激反应和分化。新陈代谢和其他细胞通路之间的串扰的重要性是显而易见的，而且越来越明显的是，人类过多的疾病与新陈代谢和其他细胞通路信号之间的界面调节不当直接相关。尽管了解新陈代谢如何与其他细胞过程沟通很重要，但我们在分子水平上的了解充其量也是非常有限的。这项提案的母方拨款使用了一条将含硫氨基酸代谢与细胞增殖联系起来的途径作为模型，以了解非蛋白水解性泛素化的调控。其核心是泛素连接酶SCFMet30，它将含硫代谢物的代谢与细胞周期结合在一起。这一修订申请建议显著扩大父母赠款的范围，以了解含硫代谢物水平如何调节SCFMet30泛素连接酶(特定目标1)，并探索泛素连接酶直接连接代谢途径与其他细胞过程的假设(特定目标2)。我们将开发和应用质谱学方法来探索代谢产物与SCFMet30途径的组件(特定目标1)以及酵母和人类细胞中的其他泛素连接酶(特定目标2)的相互作用。然后，将使用已识别结合部位的突变来探索已识别的相互作用的生理重要性。泛素连接酶是种类最多的一类细胞调节剂，在植物系统中令人兴奋的发现表明泛素连接酶是代谢物相关小分子的受体。这项提议旨在将泛素连接酶定义为酵母和人类新陈代谢和其他细胞过程之间的分子联系。这些研究的发现可能定义代谢物感知的新范式，并发现与疾病相关的新途径。

DESCRIPTION (provided by applicant Protein modification with the small protein ubiquitin, a process referred to as ubiquitylation, plays crucial roles in the majority of cellular processes. Ubiquitin ligases are the most complex and most important components of the ubiquitylation machinery. They confer substrate selectivity and are the main targets for regulation. The importance of ubiquitin ligases (E3 enzymes) is underscored by the vast number of proteins functioning as E3s. It is estimated that the human genome encodes 600- 1000 proteins with ubiquitin ligase activity, a number that is significantly higher than that of protein kinases. Ubiquitin ligases are poised to form a link between metabolic pathways and other cellular processes such as cell cycle, stress response, and differentiation. The importance of cross talk between metabolism and other cellular pathways is evident, and it is becoming increasingly clear that a plethora of human diseases is directly connected to misregulation at the interface between metabolism and signaling to other cellular pathways. Despite the importance of understanding how metabolism communicates with other cellular processes, our understanding at the molecular level is very limited at best. The parent grant of this proposal uses a pathway that connects sulfur amino acid metabolism with cell proliferation as a model to understand regulation by non-proteolytic ubiquitylation. The central player is the ubiquitin ligase SCFMet30, which integrates metabolism of sulfur containing metabolites with the cell cycle. This revision application proposes to significantly extend the scope of the parent grant to understand how levels of sulfur containing metabolites regulate the SCFMet30 ubiquitin ligase (specific aim 1), and to explore the hypothesis that ubiquitin ligases directly connect metabolic pathways with other cellular processes (specific aim 2). We will develop and apply mass spectrometric approaches to probe interactions of metabolites with components of the SCFMet30 pathway (specific aim 1), and other ubiquitin ligases in yeast and human cells (specific aim 2). The physiological importance of identified interactions will then be probed using mutations in the identified binding sites. Ubiquitin ligases are the most diverse group of cellular regulators and exciting findings in the plant system have demonstrated ubiquitin ligases as receptors for metabolite related small molecules. This proposal aims to define ubiquitin ligases as the molecular link between metabolism and other cellular processes in yeast and humans. Findings from these studies are likely to define new paradigms in metabolite sensing and to uncover novel disease related pathways.