An investigation into the CFTR-Associated Ligand (CAL) and the underlying molecular mechanism by which it increases cell surface expression of ΔF508-CFTR through regulation of trafficking pathways

描述(由申请人提供)：囊性纤维化跨膜电导调节因子(CFTR)的突变通过影响肺部和其他几个关键器官的水/盐平衡，导致囊性纤维化，这是一种常见的致命性常染色体隐性遗传病。Cftr相关配体(CAL)是一种与cftr C末端结合的PDZ结构域结合蛋白。虽然以前已经证明CAL减少了细胞表面的WT-cftr并促进了它在溶酶体中的降解，但cal与ΔF508-cftr的相互作用从未被表征过。这项研究的目的是阐明CAL如何调控ΔF508-cftr的贩运和降解。目前的建议认为，CAL对ΔF508-cftr突变体的ER和ER后转运至关重要，可能是患者值得治疗的靶点。该提议基于两个特定的目的，第一个目的是阐明CA1在ΔF508-CFtR早期运输途径中的生理作用。第二个目标是确定在ΔF508-cftr调控过程中，哪些辅助蛋白，包括伴侣蛋白和众所周知的CAL相互作用蛋白，受到CAL的影响或与CAL结合。初步数据表明，CAL增加了ΔF508-cftr在细胞表面的表达，并在蛋白酶体中被降解。研究型培训计划的设计是为了在两年内成功完成所述目标，因为学生已经完成了所有其他必要的学位计划要求，并且已经有了令人振奋的初步数据。实现所述研究目标的方法集中在细胞培养工作和显微镜检查上，这两项工作在约翰霍普金斯大学医学院内都很容易获得，因此在范围上是现实的。

DESCRIPTION (provided by applicant): Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) cause Cystic Fibrosis, a common lethal autosomal recessive disorder, by affecting the water/salt balance of the lungs and several other key organs. The CFTR-Associated Ligand (CAL) is a PDZ domain binding protein that binds to the C-terminus of CFTR. While it has been previously shown that CAL reduces cell surface WT-CFTR and promotes its degradation in the lysosome, CAL's interactions with ΔF508-CFTR have never been characterized. The goal of this study is to illuminate how CAL regulates trafficking and degradation of ΔF508-CFTR. The present proposal posits that CAL is critical for ER and post-ER trafficking of the ΔF508-CFTR mutant and may be a worthwhile target for treatment in patients. The proposal is built upon two specific aims, the first of which is to elucidate to the physiological role of CAL in the early trafficking pathway of ΔF508-CFTR. The second aim is focused on determining which accessory proteins, including chaperones and well known CAL interacting proteins, are influenced by or bound to CAL in the process of ΔF508-CFTR regulation. Preliminary data has shown that CAL increases cell surface expression of ΔF508-CFTR and is degraded in the proteasome. The research training program has been designed for successful completion of the stated aims in two years, as the student has completed all other necessary degree program requirements and already has promising preliminary data. Methods for achieving the stated goals of the research are focused on cell culture work and microscopy, both of which are easily accessible within the Johns Hopkins School of Medicine and thus are realistic in scope.