Molecular Analysis of Multivesicular Body Formation

描述（由申请方提供）：内体将预定在溶酶体中降解的内吞大分子与再循环回细胞表面或路由至其他细胞内细胞器的分子分离。多泡体（MVB）是一种晚期内体，含有内体膜向腔室内陷形成的囊泡。许多生长因子受体和免疫系统的刺激成分被分选到MVB囊泡中。此外，人类免疫缺陷病毒-1和其他包膜RNA病毒篡夺MVB囊泡形成所需的组分以产生病毒体，从而使病毒感染得以传播。本申请的目的是确定MVB囊泡介导的转运的分子基础。该项目的先前工作已经确定了酿酒酵母中Bro 1蛋白参与MVB货物分选途径的机制。拟议的工作旨在扩大这种分析的范围，以了解调节MVB囊泡形成的分子机制。在缺乏转运所需的功能性内体分选复合物（ESCRT）的细胞中，由于内体形成“E类隔室”，该过程受损。“初步研究表明，E类区室的形成是由内体上泛素化蛋白的积累驱动的，并取决于内体束缚复合物。具体目标1将确定调节蛋白质内体去泛素化的机制，具体目标2将探索泛素化蛋白质和拴系组分如何合作创建E类隔室，具体目标3将研究调节Snf 7聚合的机制，Snf 7被认为是MVB囊泡出芽的驱动力。所有这三个具体目标将采用遗传，生物化学和显微镜研究相结合，以检查各种突变酵母菌株。 公共卫生相关性：该项目旨在了解酵母ESCRT机制的机制调节，这与在人类细胞中发挥作用的ESCRT机制高度相关。在人类中，ESCRT机制的功能是下调许多控制细胞分裂的受体激活的信号转导途径，肿瘤发生与破坏ESCRT活性的突变有关，使ESCRT机制的组分成为基因治疗的候选靶点。ESCRT机制也被人类免疫缺陷病毒-1篡夺，用于产生感染性病毒体，使ESCRT机制的组分成为抗病毒治疗的候选靶标。

DESCRIPTION (provided by applicant): Endosomes segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed to other intracellular organelles. The multivesicular body (MVB) is a late endosome that contains vesicles formed by invagination of the endosomal membrane toward the compartmental lumen. Many growth factor receptors and stimulatory components of the immune system are sorted into MVB vesicles. Furthermore, human immunodeficiency virus-1 and other enveloped RNA viruses usurp components required for MVB vesicle formation in order to produce virions, thereby enabling the spread of viral infection. The objective of this application is to determine the molecular basis of MVB vesicle-mediated transport. Previous work on this project has identified mechanisms by which the Bro1 protein in Saccharomyces cerevisiae participates in the MVB cargo sorting pathway. The proposed work seeks to expand the scope of this analysis to understand the molecular mechanisms that regulate MVB vesicle formation. This process is impaired in cells lacking functional endosomal sorting complexes required for transport (ESCRTs) as a result of endosomes forming `class E compartments.' Preliminary studies suggest that class E compartment formation is driven by the accumulation of ubiquitinated proteins at endosomes and depends on endosomal tethering complexes. Specific aim 1 will determine the mechanism that regulates protein deubiquitination at endosomes, specific aim 2 will explore how ubiquitinated proteins and tethering components cooperate to create class E compartments, and specific aim 3 will investigate the mechanism that regulates polymerization of Snf7, which has been implicated as the driving force of MVB vesicle budding. All three specific aims will be pursued using a combination of genetic, biochemical, and microscopic studies to examine a variety of mutant yeast strains. PUBLIC HEALTH RELEVANCE: The proposed project seeks to understand the mechanistic regulation of the yeast ESCRT machinery, which is highly related to the ESCRT machinery that functions in human cells. In humans, the ESCRT machinery functions to downregulate many receptor-activated signal transduction pathways that control cell division, and tumorigenesis has been linked to mutations that disrupt ESCRT activity, making components of the ESCRT machinery candidate targets for gene therapy. The ESCRT machinery also is usurped by the human immunodeficiency virus-1 for the production of infectious virions, making components of the ESCRT machinery candidate targets for anti-viral therapies.