The CFTR Interactome

项目摘要 囊性纤维化（CF）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，影响美国出生的4,000名儿童中的1名（www.cff.org）。今天，CF疾病的症状 在最常见的突变患者（CFTR 508）中，可以用三种不同的药物改善 组合。然而，这些药物具有负面的副作用，降低了患者对治疗的依从性。 并造成长期的健康风险。此外，许多其他常见的CF引起的突变， 对目前的任何CF药物都很差或根本不起作用，使携带这些突变的CF患者仅具有 对症治疗开发用于此类CF变体的新化合物的努力受到缺乏 蛋白质结构，将揭示这些变体的构象缺陷，主要是由于技术 表达和纯化足够量的这些不稳定蛋白质的困难。表征 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 先前开发的共价蛋白绘画（CPP），一种用于体内结构表征的新方法， 蛋白质质谱在这里，我们建议开发一种更灵敏和可复用的CPP方法， 命名为bioTMT-CPP，这将有助于检测和比较CFTR之间的构象变化， 样品新方法将有助于表征错误折叠的CFTR中的构象缺陷 对当前疗法难治的变体。此外，我们的方法有可能确定药物 结合位点，并确定目前CF药物的作用机制，这仍然是未知的三个 四种活性化合物。这些知识将有助于合理化药物联合治疗。我们亦建议 功能上表征了一种新的CFTR构象，我们发现了CPP，并获得了 错误折叠和失活的CFTR，可能在蛋白质运输过程中。深入了解分子机制 稳定该构象以及将其释放成活性构象那些构象对于 进一步的校正药物开发，将有利于所有CF患者。

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.