Modulation of Rab7-Dependent Degradative Pathways by Novel Adenovirus Protein

描述（由申请人提供）：内吞作用具有许多重要的功能，从获取细胞外营养物质到调节细胞表面受体表达和信号转导，维持细胞极性和抗原呈递。许多细胞内病原体劫持内吞途径，以侵入细胞，增殖，并确保病原体存活。了解致病性感染的后果使人们能够更好地了解内吞贩运机制。在人类疾病模型中，分离的致病基因也被用于抑制与其细胞靶点相关的病理过程。本提案的重点是了解由人类腺病毒早期3区编码的完整膜蛋白所采用的分子和细胞机制。它最初被发现是因为它能够将组成循环的EGF受体转移到溶酶体上。我们最近发现RID？与RILP和ORP1L相互作用，这两种已知的Rab7 GTPase的效应物控制着从早期到晚期内体再到溶酶体的运输。重要的是,摆脱?补偿Rab7的功能丧失，表明它通过协调Rab7效应物向通常被认为是早期分选核内体的室室募集来改变核内体膜动力学。据我们所知，这是DNA病毒编码的第一个通过Rab7模仿发挥作用的蛋白质。与其他小的GTP酶类似，Rab7通过在活性GTP结合状态和非活性gdp结合状态之间循环而起作用。相比之下，RID？是一种非酶的内在膜蛋白，与Rab7没有任何序列同源性，为趋同进化提供了一个显著的例子。四个具体目标将检验这些假设。1)去掉吗?通过招募RILP和ORP1L来控制微管依赖的囊泡运输，然后激活负端定向动力蛋白-动力蛋白马达。2)。掉吗?- RILP促进独立于受体酪氨酸激酶或泛素状态的escrt - ii依赖性EGF受体分选。3)消除?-ORP1L调节胆固醇从核内体流出，维持细胞内适当的脂质平衡。4)消除?在生殖腺病毒感染期间补偿Rab7的功能丧失，并通过干扰TNF?来减弱腺病毒诱导的炎症性疾病。-EGF受体信号级联调节IL-8的产生。总之，这些研究将为膜蛋白运输的细胞生物学提供新的见解，也为腺病毒诱导的炎症疾病的分子基础提供新的见解。拟议的研究与公共卫生相关，因为它们将为腺病毒肺炎的分子发病机制提供新的见解。它们将产生设计无毒腺病毒基因治疗载体的新策略，并使慢性阻塞性肺疾病（COPD）和其他慢性呼吸道疾病患者受益，在这些疾病中，持续腺病毒感染被认为是一个重要的疾病风险因素。这些研究还将为开发针对尼曼-匹克C病（一种总是致命的进行性神经系统疾病）患者的新疗法提供基础。

DESCRIPTION (provided by applicant): Endocytosis serves many important functions ranging from acquisition of extracellular nutrients to regulation of cell surface receptor expression and signal transduction, maintenance of cell polarity, and antigen presentation. Many intracellular pathogens hijack endocytic pathways in order to invade cells, proliferate, and ensure pathogen survival. Understanding the consequences of pathogenic infection has enabled greater understanding of the endocytic trafficking machinery. Isolated pathogenic genes have also been used to curb pathological processes associated with their cellular targets in human disease models. The focus of this proposal is to understand the molecular and cellular mechanisms employed by an integral membrane protein encoded by the early region 3 of human adenoviruses called RID?, which was originally identified because of its ability to divert constitutively recycling EGF receptors to lysosomes. We have recently discovered that RID? interacts with RILP and ORP1L, two known effectors for the Rab7 GTPase that governs transport from early to late endosomes and then to lysosomes. Importantly, RID? compensates for Rab7 loss-of-function, suggesting it modifies endosome membrane dynamics by coordinating recruitment of Rab7 effectors to compartments that would ordinarily be perceived as early sorting endosomes. To our knowledge this is the first protein encoded by a DNA virus that functions by Rab7 mimicry. Similar to other small GTPases, Rab7 acts by cycling between an active GTP- bound state and an inactive GDP-bound state. In contrast RID? is a non-enzymatic intrinsic membrane protein that lacks any sequence homology to Rab7, providing a remarkable example of convergent evolution. Four specific aims will test these hypotheses. 1) RID? controls microtubule-dependent vesicle transport by recruiting RILP and ORP1L which then activate minus end-directed dynein-dynactin motors. 2). RID?- RILP facilitates ESCRT-II-dependent EGF receptor sorting independent of receptor tyrosine kinase or ubiquitin status. 3) RID?-ORP1L regulates cholesterol efflux from endosomes necessary to maintain the proper lipid balance in cells. 4) RID? compensates for Rab7 loss-of-function during a productive adenovirus infection, and blunts adenovirus-induced inflammatory disease by interfering with a TNF?-EGF receptor signaling cascade that regulates IL-8 production. Altogether these studies will provide novel insights to the cell biology of membrane protein trafficking, and also the molecular basis for adenovirus-induced inflammatory disease. PUBLIC HEALTH RELEVANCE The proposed studies are relevant to public health because they will provide novel insights to the molecular pathogenesis of adenovirus pneumonia. They will generate new strategies for design of non-toxic adenovirus-based gene therapy vectors, and benefit patients with chronic obstructive pulmonary disease (COPD) and other chronic respiratory diseases where it is thought persistent adenovirus infections are an important disease risk factor. These studies will also provide the foundation for developing new treatments for patients with Niemann-Pick C disease, a progressive neurological disease that is always fatal.