Discovery of Novel Mechanisms of Action of Ubiquitin-Like Proteins in Cellular Stress Pathways

项目摘要 真核细胞应激反应是高度保守和精确调控的。当这些途径 功能障碍通常会导致严重的生理后果，例如癌症， 神经变性疾病和自身免疫性疾病。泛素样修饰（UBLs）是快速、可逆的， 并能深刻地改变细胞的命运和功能。有趣的是，大多数UBL参与细胞内的 对应激的反应，特别是对感染、ER应激和自噬的反应。类泛素蛋白 泛素结构保守性以及共价修饰的靶蛋白，然而UBL具有极其 不同的功能，其范围从转录抑制到自噬启动。与泛素不同， ISG化或ATG12化对靶蛋白命运和功能的影响在很大程度上是未知的。在这 我们的目标是解码真核生物应激反应后细胞的翻译后景观 使用复杂的蛋白质组学策略来解决ISG15和ATG12如何改变的基本问题， 它们的共价靶细胞的功能。 对于ISG15，我的实验室最近的工作确定了它在控制宿主代谢过程中的新作用， 该提案解决了mTOR和其他关键上游调节因子的ISGylation如何分解代谢过程， 自噬调节细胞和动物的代谢和分解代谢能力。 对于ATG12，我们开创了一种结合尖端蛋白质组学的体内遗传方法， 自噬相关的泛素样蛋白ATG 12的内源性底物诱导细胞凋亡后， 应激途径如线粒体外膜透化或内质网应激。

PROJECT SUMMARY Eukaryotic cellular stress responses are highly conserved and precisely regulated. When these pathways malfunction there are often grave physiological consequences in the form of diseases such as cancer, neurodegenerative disease and autoimmune disorders. Ubiquitin-like modifications (UBLs) are rapid, reversible and can profoundly alter cell fate and function. Intriguingly, the majority of UBLs are involved in the cellular response to stress, in particular the response to infection, ER-stress and autophagy. Ubiquitin-like proteins share structural conservation with ubiquitin and also covalently modify target proteins, however UBLs have extremely divergent functions, which range from transcriptional repression to autophagy initiation. Unlike ubiquitin, the consequences of ISGylation or ATG12ylation on target protein fate and function are largely unknown. In this proposal, we aim to decode the post-translational landscape of the cell following eukaryotic stress responses using sophisticated proteomics strategies to address the fundamental question of how ISG15 and ATG12 alter the function of their covalent cellular targets. For ISG15, my laboratory’s recent work identified a novel role in the control of host metabolic processes and this proposal addresses how ISGylation of mTOR and other key upstream regulators of the catabolic process of autophagy tunes the metabolic and catabolic capacity of cells and animals. For ATG12, we have pioneered an in vivo genetic method coupled with cutting-edge proteomics to identify endogenous substrates of the autophagy-related ubiquitin-like protein ATG12 following induction of cellular stress pathways such as mitochondrial outer membrane permeabilization or endoplasmic reticulum stress.