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（由申请方提供）：囊性纤维化跨膜传导调节因子（CFTR）突变通过影响肺和其他几个关键器官的水/盐平衡引起囊性纤维化，这是一种常见的致死性常染色体隐性遗传病。CFTR相关配体（CAL）是一种PDZ结构域结合蛋白，与CFTR的C-末端结合。虽然先前已经表明CAL降低细胞表面WT-CFTR并促进其在溶酶体中的降解，但CAL与ΔF508-CFTR的相互作用从未被表征。本研究的目的是阐明CAL如何调节ΔF508-CFTR的运输和降解。目前的提议假定CAL对于ΔF508-CFTR突变体的ER和ER后运输至关重要，并且可能是患者中值得治疗的靶标。该提议基于两个具体目标，第一个是阐明CAL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调节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ΔF508-CFTR的细胞表面表达，并在蛋白酶体中降解。研究培训计划旨在在两年内成功完成既定目标，因为学生已经完成了所有其他必要的学位课程要求，并且已经拥有有希望的初步数据。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，这两种方法在约翰霍普金斯医学院都很容易获得，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.