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

小窝蛋白-1(CAV1)及其异构体是小窝的主要成分，在细胞机械保护、内吞作用和信号转导中发挥重要作用。CAV1的单一泛素化控制着CAV1沿内吞途径的运输，并分选到内溶体中进行降解。泛素化CAV1的一个效应因子(Ub-CAV1)是VCP/p97AAA-ATPase的内体复合体。至关重要的是，VCP/p97的疾病相关突变会导致人类肌肉和神经元退化，影响Ub-CAV1结合和内体分选。然而，该过程的关键调控因子，如CAV1特异的泛素连接酶和Ub-CAV1的其他下游效应因子尚不清楚。利用基于显微镜的泛素化实验，我们希望进行siRNA介导的筛选，以确定相关的E3泛素连接酶。同时，我们希望进行CAV1亲和纯化与质谱分析相结合，以鉴定更多介导Ub-CAV1转运的效应蛋白。利用已建立的组织培养细胞转运试验，我们将研究这些因素如何在内体途径中介导Ub-CAV1分选的不同步骤，以及在溶酶体中如何调节CAV1的周转。预期的结果将有助于理解CAV1的一个新的调节层，并为进一步深入了解由p97突变和内溶酶系统相关缺陷引起的退行性疾病的复杂发病机制。

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.