Regulation of Nutrient Homeostasis by COMMD proteins

质膜蛋白，包括受体和营养转运体，经历内化进入内质体室，在那里这些蛋白被循环回到质膜。这一内体循环过程对细胞内环境的稳定至关重要。我们的实验室发现了两个相互关联的复合体，它们在内体循环中发挥关键作用，即CCC和Retriver。这两个组件都与WASH协同工作，WASH是一种内体定位的肌动蛋白核化五聚体复合体。值得注意的是，这些系统的扰动具有深远的后果，包括发育异常、铜和脂质处理改变以及糖耐量缺陷。这些生理变化可以追溯到关键受体和转运体的错误运输，包括Notch、ATP7A/ATP7B、LDLR和GLUT2。在其核心，CCC复合体含有COMMD蛋白，与两个卷曲蛋白CCDC22和CCDC93结合在一起。我们最近证明，CCC通过与PI(3)P磷酸酶MTMR2相互作用来限制内体PI(3)P的数量，从而调节WASH复合体。我们还发现，CCC复合体招募了一个名为寻回犬的货物识别复合体，其功能是识别需要回收的蛋白质。虽然我们在剖析这些系统的机械基础方面取得了很大进展，但我们的理解仍然是初级的。这个项目的总体目标是提供一幅CCC介导的洗涤和寻回调控的深层机制图，将我们的注意力集中在营养调节剂的内体循环上。基于新出现的数据，这一建议将检验这一假设，即CCC复合体协调PI(3)P水平和Rab21激活，以促进检索器介导的表面蛋白的循环。本项目将集中于以下具体目标：(1)确定CCC调节MTMR对内体作用的机制。为此，我们将研究CCC复合体如何协调MTMR的行动，特别是通过MTMR5，以及Rab21的激活和寻回犬的招募。(2)明确PI(3)P调节WASH复合体募集和活性的机制。在这一目标中，我们将确定FAM21和WASH中PI(3)P结合结构域调节内体招募和WASH复合体活性的机制，以及这些活动最终如何影响内体转运。(3)研究体内货物蛋白对寻回犬的依赖调节。在这个目的中，我们将利用新产生的寻回犬缺陷小鼠模型(条件性Vps35l，Vps26c等位基因)来检查该系统对ATP7B和LDLR在肝脏中运输的贡献。此外，我们将利用蛋白质组学来确定在哺乳动物肝脏中受检索器、CCC和WASH调控的质膜蛋白的宽度。总而言之，这个项目将阐明管理横穿内体系统的无数货物贩运的重要原则。放松对这些途径的管制对正常的生物/细胞生理学有广泛的影响，因此它的科学影响将对几个生物医学领域产生深远的影响。

Plasma membrane proteins, including receptors and nutrient transporters, undergo internalization into the endosomal compartment, from where these proteins are recycled back to the plasma membrane. This endosomal recycling process is essential to cellular homeostasis. Our laboratories have discovered two interrelated complexes that play key roles in endosomal recycling known as CCC and retriever. Both of these assemblies work in concert with WASH, an endosomally-localized actin nucleating pentameric complex. Significantly, perturbations of these systems have far-reaching consequences, including developmental anomalies, altered copper and lipid handling, and defective glucose tolerance. These physiologic alterations can be traced to faulty trafficking of key receptors and transporters including Notch, ATP7A/ATP7B, LDLR and GLUT2. At its core, the CCC complex contains COMMD proteins, in association with two coiled-coil proteins, CCDC22 and CCDC93. We recently demonstrated that CCC regulates the WASH complex by limiting the amount of endosomal PI(3)P through an interaction with the PI(3)P phosphatase MTMR2. We also uncovered that the CCC complex recruits a cargo recognition complex, termed retriever, whose function is to identify proteins that need to be recycled. While we made great progress in dissecting the mechanistic underpinnings of these systems, our understanding is still rudimentary. The overall goal of this project is to provide a deep mechanistic picture of CCC-mediated regulation of WASH and retriever, focusing our attention on endosomal recycling of nutrient regulators. Based on new emerging data, this proposal will test the hypothesis that the CCC complex coordinates both PI(3)P levels and Rab21 activation to promote retriever-mediated recycling of surface proteins. This project will focus on the following specific aims: (1) Determine the mechanism by which CCC regulates MTMR actions on endosomes. In this aim we will examine how the CCC complex coordinates MTMR actions, particularly by MTMR5, together with activation of Rab21 and retriever recruitment. (2) Define the mechanism by which PI(3)P regulates the recruitment and activity of the WASH complex. In this aim, we will define the mechanisms by which PI(3)P-binding domains in FAM21 and WASH regulate the endosomal recruitment and activity of the WASH complex, and how these activities ultimately impact endosomal trafficking. (3) Examine retriever-dependent regulation of cargo proteins in vivo. In this aim we will utilize newly generated mouse models of retriever deficiency (conditional Vps35l, Vps26c alleles) to examine the contribution of this system to ATP7B and LDLR trafficking in the liver. Furthermore, we will utilize proteomics to define the breadth of plasma membrane proteins regulated by retriever, CCC, and WASH in the mammalian liver. Altogether, this project will elucidate important principles that govern the trafficking of a myriad of cargoes that traverse the endosomal system. Deregulation of these pathways have a broad impact on normal organismal/cellular physiology, thus its scientific impact will have far reaching implications for several biomedical fields.