Molecular Analysis of Multivesicular Body Formation

多泡体形成的分子分析

• 批准号: 7924952
• 负责人: CHARLES G ODORIZZI
• 金额: $ 24.62万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924952
• 关键词: Adopted; Back; Binding; Biochemical; Biogenesis; Cell division; Cell surface; Cells; Complement; Complex; Deubiquitination; Endocytosis; Endosomes; Genes; Genetic; Growth Factor Receptors; HIV-1; Human; Hydrolase; Immune system; Integral Membrane Protein; Link; Lysosomes; Mediating; Membrane; Microscopic; Molecular; Molecular Analysis; Morphology; Multivesicular Body; Mutation; Organelles; Pathway interactions; Process; Production; Proteins; Publishing; RNA Viruses; Recruitment Activity; Recycling; Regulation; Research; Role; Route; Saccharomyces cerevisiae; Signal Transduction Pathway; Sorting - Cell Movement; Ubiquitin; Vesicle; Viral; Virion; Virus Diseases; Work; Yeasts; base; driving force; gene therapy; late endosome; macromolecule; mutant; overexpression; polymerization; public health relevance; receptor; tumorigenesis; unpublished works

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.

描述（由申请人提供）：内体隔离内吞大的大分子，这些大分子注定要从分子中降解在溶酶体中，这些分子被回收回到细胞表面，要么被路由到其他细胞内细胞器。多囊体（MVB）是一个晚期内体，其中包含通过内体膜向室腔的内体膜形成的囊泡。免疫系统的许多生长因子受体和刺激成分被分类为MVB囊泡。此外，人类免疫缺陷病毒-1和其他包膜RNA病毒篡夺了MVB囊泡形成所需的成分，以产生病毒体，从而实现病毒感染的传播。该应用的目的是确定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的Bro1蛋白参与MVB货物分类途径的机制。拟议的工作旨在扩大该分析的范围，以了解调节MVB囊泡形成的分子机制。由于内体形成“ E类隔室”所需的运输（ESCRT）所需的功能性内体分类复合物（ESCRTS）所需的功能性内体分选复合物的细胞受损。初步研究表明，E类室的形成是由泛素化蛋白在内体上的积累驱动的，并取决于内体束缚复合物。具体目标1将确定调节内体蛋白质去泛素化的机制，具体目标2将探讨如何配合使用泛素化的蛋白质和绑扎组件来创建E类隔室，而特定的目标3将调查调节SNF7聚合的机制，该机制已被视为MVB Vesicle butding的驱动力。将使用遗传，生化和微观研究的组合来追求这三个特定目标，以检查各种突变的酵母菌菌株。 公共卫生相关性：拟议的项目旨在了解酵母机械机械的机械调节，该机械与在人类细胞中起作用的ESCRT机械高度相关。在人类中，ESCRT机械的功能下调了许多受体激活的信号转导途径，控制细胞分裂，肿瘤发生与破坏ESCRT活性的突变有关，从而使ESCRT机械候选靶标的组成部分用于基因治疗。人类免疫缺陷病毒1也篡夺了抗病毒疗法的ESCRT机械候选靶标的组成部分。