Regulation of WASH-mediated Endosomal Protein Recycling

描述（由申请人提供）：蛋白质在整个细胞中的运输是一个高度调控的过程，是细胞生物学的一个基本方面。内体蛋白回收途径是一个重要的细胞运输过程，它通过将溶酶体中降解的蛋白质从内体回收回高尔基体或质膜，从而促进蛋白质的修复。这一基本细胞过程的主要贡献者是WASH蛋白。WASH是肌动蛋白成核促进因子WASP家族的一员，专门促进肌动蛋白在核内体上的组装，促进蛋白质循环。重要的是，WASH和内体蛋白循环与许多人类疾病有关，包括遗传性散发性截瘫、阿尔茨海默氏症和癌症。此外，这种细胞过程是将霍乱、蓖麻毒素和炭疽等致命致病性毒素运送到其作用部位所必需的。因此，了解WASH的功能及其调控对人类健康具有重要意义。由于其与多亚基复合物（称为WASH调控复合物或SHRC）的结合，WASH处于自动抑制状态。最近，有报道称WASH是由泛素化激活的。泛素化是一种翻译后修饰，可以改变细胞中蛋白质的稳定性、确认性和/或活性。由MAGE-L2-TRIM27 E3泛素连接酶介导的WASH非典型k63连锁泛素化是WASH激活、内体f -肌动蛋白生成和内体蛋白循环的重要事件。利用多种体外生化和细胞方法，本项目旨在通过翻译后修饰确定调节WASH活性的机制和信号通路。此外，本研究旨在确定WASH和MAGE-L2-TRIM27的生理功能，重点研究它们的疾病相关功能。这些研究对于我们理解wash介导的内体蛋白循环具有重要意义，打破了与操纵这一细胞过程相关的人类疾病治疗的关键障碍，并推进了我们对这一基本细胞过程的科学知识。

DESCRIPTION (provided by applicant): Transport of proteins throughout the cell is a highly regulated process that is a fundamental aspect of cell biology. The endosomal protein recycling pathway is one important cellular transport process which promotes the rescue of proteins destined for degradation in the lysosome by recycling them back to the Golgi or plasma membrane from endosomes. A major contributor to this fundamental cellular process is the WASH protein. WASH is part of the WASP family of actin nucleation promoting factors and specifically promotes actin assembly on endosomes to facilitate protein recycling. Importantly, WASH and endosomal protein recycling have been linked to a number of human diseases, including hereditary sporadic paraplegia, Alzheimer's, and cancer. Furthermore, this cellular process is required for the transport of lethal pathogenic toxins, such as cholera, ricin, and anthrax to their site of action. Therefore, understanding the function and regulation of WASH has important implication on human health. WASH is held in an auto-inhibited state due to its incorporation into a multi-subunit complex, referred to as the WASH regulatory complex or SHRC. Recently, it was described that WASH is activated by ubiquitination. Ubiquitination is a post-translational modification that can alter the stability, confirmation, and/or activity of protins in cells. Non-canonical K63-linked ubiquitination of WASH by the MAGE-L2-TRIM27 E3 ubiquitin ligase is an essential event in WASH activation, generation of endosomal F-actin, and endosomal protein recycling. Utilizing a number of in vitro biochemical and cellular approaches, this project aims to determine the mechanisms and signaling pathways regulating WASH activity through post-translational modifications. Furthermore, it aims to determine the physiological functions of WASH and MAGE-L2-TRIM27 with emphasis on their disease-related functions. These studies are important for our understanding of WASH-mediated endosomal protein recycling, breaking critical barriers to treating human diseases associated with manipulation of this cellular process, and advancing our scientific knowledge of this fundamental cellular process.