Ubiquitin-regulated sorting of Caveolin-1 in the endosomal system

内体系统中 Caveolin-1 的泛素调节分选

• 批准号: 272946205
• 负责人: Professor Dr. Hemmo Meyer
• 金额: --
• 依托单位: Abteilung Molekularbiologie I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/272946205?language=en
• 关键词: Ubiquitin; regulated; sorting; Caveolin; endosomal

Caveolin-1 (CAV1) and its isoforms are the major constituents of caveolae, which play important roles in cellular mechano-protection, endocytosis, and signalling. Mono-ubiquitination of CAV1 governs CAV1 trafficking along the endocytic pathway and sorting into endolysosomes for degradation. One effector of ubiquitinated CAV1 (Ub-CAV1) is an endosomal complex of the VCP/p97 AAA-ATPase. Crucially, disease-associated mutations in VCP/p97, which cause muscle and neuronal degeneration in humans, affect Ub-CAV1 binding and endosomal sorting. However, key regulators of the process such as the CAV1-specific ubiquitin ligase and additional downstream effectors of Ub-CAV1 are unknown. Using a microscopy-based ubiquitination assay, we wish to carry out an siRNA-mediated screen to identify the relevant E3 ubiquitin ligase. In parallel, we wish to perform CAV1 affinity purification coupled with mass spectrometry to identify additional effector proteins that mediate transport of Ub-CAV1. Using established transport assays in tissue culture cells, we will study how these factors mediate different steps in Ub-CAV1 sorting in endosomal pathways and CAV1 turnover in the lysosome. The expected results will help understand a novel layer of regulation of CAV1, and provide further insights into the complex pathogenesis of degenerative diseases caused by mutations in p97 and related defects in the endolysosomal system.

Caveolin-1（CAV 1）及其亚型是Caveolae的主要组成部分，在细胞的机械保护、内吞和信号转导中发挥重要作用。CAV 1的单泛素化控制CAV 1沿着内吞途径运输并分选到内溶酶体中进行降解。泛素化CAV 1（Ub-CAV 1）的一个效应子是VCP/p97 AAA-ATP酶的内体复合物。至关重要的是，VCP/p97中的疾病相关突变会导致人类肌肉和神经元变性，影响Ub-CAV 1结合和内体分选。然而，该过程的关键调控因子，如CAV 1特异性泛素连接酶和Ub-CAV 1的其他下游效应因子尚不清楚。使用显微镜下的泛素化测定，我们希望进行siRNA介导的筛选，以确定相关的E3泛素连接酶。与此同时，我们希望进行CAV 1亲和纯化与质谱联用，以鉴定介导Ub-CAV 1转运的其他效应蛋白。使用组织培养细胞中已建立的转运试验，我们将研究这些因素如何介导Ub-CAV 1内体途径中的分选和溶酶体中的CAV 1周转的不同步骤。预期的结果将有助于了解一个新的层的调节CAV 1，并提供进一步的见解p97突变和相关缺陷的内溶酶体系统引起的退行性疾病的复杂发病机制。