Chaperones and membrane protein biogenesis

描述(申请人提供)：拟议工作的目标是确定胞质分子伴侣蛋白如何在蛋白质质量控制中发挥作用，以促进膜蛋白的折叠和降解。解决这一问题的途径是研究囊性纤维化跨膜电导调节因子(CFTR)的正常和致病突变形式的生物发生。我们之前利用培养细胞和无细胞系统进行的研究明确了胞质Hsc70伴侣系统在促进新生内质网(ER)形式CFTR的折叠和降解中的作用。上一个资助期产生的数据表明，Hsc70在含有内质网本地化Hsp40 HDJ-2的复合体中发挥作用，以促进CFTR折叠/组装。此外，我们发现Hsc70与一种名为CHIP的新型辅助伴侣相互作用，该CHIP含有名为U-box的E3泛素连接酶结构域，以促进CFTR泛素化和降解。这些数据表明，胞质Hsc70在膜蛋白的折叠和降解过程中都发挥着作用，并为辅助伴侣有助于调节新生膜蛋白在折叠和降解途径之间的分配提供了证据。我们现在寻求更多的资金来扩展这些研究，并提出一系列实验，旨在进一步阐明CFTR折叠和降解的途径。这些研究将在三个具体目标下进行。目的1.研究胞浆和腔内ER伴侣系统在CFTR折叠中的作用。目的2.确定Hsp70/CHIP复合体控制新生cftr在折叠和降解途径之间分配的机制。目的3.确定促进泛素化CFTR递送到蛋白酶体的成分。总体而言，这些研究将提供一个全面的观点，即细胞如何调节CFTR的折叠和降解。这些研究的长期目标是提供信息，以确定开发治疗CF的靶点。

DESCRIPTION (provided by applicant): The objective of the proposed work is to define how cytosolic molecular chaperone proteins function in protein quality control to facilitate the folding and degradation of membrane proteins. The approach to taken to solve this problem is to study the biogenesis of normal and disease causing mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR). Our previous studies which utilized cultured cells and cell free systems defined roles for the cytosolic Hsc70 chaperone system in facilitating both the folding and degradation of nascent endoplasmic reticulum (ER) forms of CFTR. Data generated in the last funding period indicate that Hsc70 acts in complex that contains the ER localized Hsp40 Hdj-2 to promote CFTR folding/assembly. In addition, we found Hsc70 to interact with a new type of co-chaperone termed CHIP that contains an E3 ubiquitin ligase domain termed the U-box, to facilitate CFTR ubiquitination and degradation. These data demonstrate that cytosolic Hsc70 functions in both the folding and degradation of membrane proteins and provide evidence that co-chaperones help mediate the partitioning of nascent membrane proteins between folding and degradation pathways. We now seek additional funding to extend these studies and propose a series of experiments that are designed to further elucidate the pathways for CFTR folding and degradation. These studies will be carried out in 3 specific aims. Aim 1. Investigate roles that cytosolic and lumenal ER chaperones systems play in CFTR folding. Aim 2. Determination of the mechanism by which the Hsp70/CHIP complex controls the partitioning of nascent CFTR between folding and degradation pathways. Aim 3. Identify the components that facilitate the delivery of ubiquitinated CFTR to the proteasome. Overall, these studies will provide a comprehensive view of how the cell mediates the folding and degradation of CFTR. The long term objective of these studies is to provide information that can identify targets for development of therapeutic to treat CF.