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功能，从获取细胞外营养素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。了解病原体感染的后果，使更多的了解内吞贩运机制。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。该提案的重点是了解由人腺病毒早期区域3编码的整合膜蛋白（称为RID？）所采用的分子和细胞机制，其最初被鉴定是因为其能够将组成性再循环的EGF受体转移到溶酶体。我们最近发现，RID？与RILP和ORP 1 L相互作用，RILP和ORP 1 L是Rab 7 GTP酶的两种已知效应物，其控制从早期到晚期内体然后到溶酶体的转运。重要的是，？补偿Rab 7的功能丧失，表明它通过协调Rab 7效应物向通常被认为是早期分选内体的区室的募集来改变内体膜动力学。据我们所知，这是第一个由DNA病毒编码的蛋白质，通过Rab 7模拟发挥作用。与其他小GTP酶类似，Rab 7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。与此同时，Rid？是一种与Rab 7缺乏任何序列同源性的非酶内在膜蛋白，提供了趋同进化的一个显著例子。四个具体目标将检验这些假设。1)摆脱？通过募集RILP和ORP 1 L控制微管依赖性囊泡运输，然后激活负末端定向动力蛋白-动力蛋白马达。2）。里德？- RILP促进ESCRT-II依赖性EGF受体分选，独立于受体酪氨酸激酶或泛素状态。3)里德？- ORP 1 L调节胆固醇从内体流出，这是维持细胞内适当脂质平衡所必需的。4)摆脱？在腺病毒感染过程中补偿Rab 7的功能丧失，并通过干扰TNF？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以及腺病毒诱导的炎症性疾病的分子基础提供新的见解。公共卫生相关性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。他们将为设计无毒的基于腺病毒的基因治疗载体产生新的策略，并使慢性阻塞性肺疾病（COPD）和其他慢性呼吸系统疾病患者受益，其中持续的腺病毒感染被认为是一个重要的疾病风险因素。这些研究还将为尼曼匹克C病患者开发新的治疗方法提供基础，尼曼匹克C病是一种进行性神经系统疾病，总是致命的。

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.