The CFTR Interactome

项目摘要 囊性纤维化是由CFTR基因突变引起的，是最常见的遗传性疾病之一 儿童疾病，影响在美国出生的4000名儿童中的1名(www.cff.org)。今天，CF病的症状 在最常见的突变(∆F508)患者中，可以用三种不同的药物改善 组合。然而，这些药物有负面副作用，会降低患者对治疗的依从性。 并构成长期的健康风险。此外，许多其他常见的导致CF的突变会对 对目前任何一种CF药物的疗效很差或根本没有，使携带这些突变的CF患者只患上 对症治疗。为这类CF变体开发新化合物的努力因缺乏 揭示这些变体的构象缺陷的蛋白质结构，主要是由于技术上的 在表达和纯化足够数量的这些不稳定蛋白方面存在困难。要刻画 错误折叠的cftr变体的构象缺陷，并帮助开发新的治疗方法，我们 共价蛋白质绘画(CPP)是一种用于体内结构表征的新方法 蛋白质的质谱学分析。在这里，我们建议开发一种更灵敏和可复用的CPP方法， 命名为BioTMT-CPP，将有助于检测和比较 样本。新的方法将有助于表征错误折叠的CFTR的构象缺陷 对目前的治疗方法难以奏效的变种。此外，我们的方法有可能精确定位药物 结合位点并确定当前CF药物的作用机制，这些药物在三种情况下仍不清楚 四种活性化合物。这些知识将有助于使药物联合疗法合理化。我们还提议 为了从功能上表征我们由CPP发现的一种新的CFTR构象，该构象是通过 错误折叠和无效的cftr，可能是在蛋白质运输过程中。洞察分子机制 稳定这种构象以及那些将其释放到活性构象的构象对于 进一步开发矫正药物，并将使所有CF患者受益。

Project Summary Cystic Fibrosis (CF) is caused by mutation of the CFTR gene and is one of the most common inherited childhood diseases, impacting 1 in 4,000 children born in the US (www.cff.org). Today, CF disease symptoms in patients with the most common mutation (∆F508 CFTR) can be improved with three different drug combinations. However, the drugs have negative side effects that reduce patient compliance to therapeutic regimens and pose long-term health risks. Additionally, many other common CF-causing mutations respond poorly or not at all to any of the current CF drugs, leaving CF patients carrying these mutations only with symptomatic therapy. Efforts to develop new compounds for such CF variants are hampered by the lack of protein structures that would reveal the conformational defects of these variants, mostly due to technical difficulties in expressing and purifying sufficient quantities of these unstable proteins. To characterize the conformational defects of misfolded CFTR variants and to aid in the development of new therapies, we previously developed Covalent Protein Painting (CPP), a novel method for in vivo structural characterization of proteins by mass spectrometry. Here, we propose to develop a more sensitive and multiplexable CPP method, named bioTMT-CPP, that will facilitate detection and comparison of CFTR conformational changes between samples. The new method will facilitate the characterization of conformational defects in misfolded CFTR variants that are refractory to current therapies. Furthermore, our approach has the potential to pinpoint drug binding sites and identify the mechanism of action of current CF drugs, which remain unknown for three of the four active compounds. Such knowledge will help to rationalize drug combination therapies. We also propose to functionally characterize a novel CFTR conformation that we discovered by CPP and that is attained by misfolded and inactive CFTR, likely during protein trafficking. Insight into the molecular mechanisms that stabilize this conformation as well as those that release it into an active conformation will be invaluable for further corrector drug development and will benefit all CF patients.