DEVELOPMENT OF MEMBRANE PROTEIN STRUCTURE

项目负责人/主要研究者（最后一名）第一，中间）：ThomaS，PhilipJ。 项目总结（见说明）： 被选中获得MERIT奖的R 01补助金的竞争性续期的原始申请 提出了三个施舍，以解决与CFTR机制相关的三个基本问题， 形成其功能性的天然结构以及这一过程如何被致病突变所改变。如何 AF 508是否会干扰NBD 1折叠？AF 508是否显著改变了 折叠NBD 1与其他域？与质量控制蛋白质的相互作用是至关重要的？期间 在MERIT支持的前四年半时间里，我们已经回答了前两个问题， 路上了这些结果表明，CFTR折叠是一个层次的过程，并提供了一个明确的解释， 校正AF 508突变体折叠的现存化合物的功效“天花板”。他们还建议， 这意味着一种基于机制的方法，用于发现与现存化合物协同作用的新化合物。 纠正剂或新的化合物，绕过“天花板”。这些方法已经在使用 通过大量的药物发现工作。最后，使用强大的特定光交联方法， 在体外翻译的蛋白质与突变型和野生型新生链的差异相互作用已经揭示 这是一种以前不受重视的先发制人的质量控制机制。该系统涉及蛋白质， 导致编码突变蛋白的mRNA的降解，从而减少 与错误折叠结合的蛋白质。这种机制可以防止潜在的细胞毒性错配蛋白的积累， 蛋白质，而不消耗能量用于无效翻译和随后的ATP依赖性蛋白水解， 蛋白酶体我们现在请求继续支持MERIT奖，以扩展分析 成功地将目标1和2中的AF 508应用于额外的CF引起突变，并定义和 描述在执行过程中发现的负责抢先质量控制系统的机制 目标3。我们感谢该研究所选择我们的研究为MERIT支持，允许追求长期的 目标3的长期发现努力，现在已经揭示了新的和意想不到的生物学。这样的道路不会 在R 01的时间限制下是可行的。 相关性（参见说明）： 囊性纤维化是一种常见的致命性遗传病，大多数病例是由干扰细胞增殖的突变引起的。 囊性纤维化跨膜传导调节因子（CFTR）蛋白的组装。建议的研究 将阐明致病突变如何干扰这一过程的细节。了解 组装过程，因此，详细的分子病理学，将提供重要信息， 开发针对囊性纤维化的靶向疗法。

Program Director/Principal Investigator (Last. First, Middle): T h o m a S , P h i l i p J . PROJECT SUMMARY (See instmctions): The original application for competitive renewal of the R01 grant that was selected for a MERIT award proposed three alms to address three fundamental questions relevant to the mechanisms by which CFTR forms Its functional, native structure and how this process Is altered by disease-causing mutations. How does AF508 interfere with NBD1 folding? Does AF508 significantly modify the interaction of the folded NBD1 with other domains? What interactions with quality control proteins are critical? During the first four and a half years of MERIT support we have answered the first two questions In an exploitable way. These results indicate that CFTR folding is a hierarchical process and provide a clear explanation for the efficacy "ceiling" for extant compounds that correct folding of the AF508 mutant. They also suggest a means to a mechanism-based approai^h for the discovery new compounds that work in synergy with extant correctors or novel compounds that circumvent the "ceiling". These approaches are already being employed by a number of drug discovery efforts. Finally, using a powerful specific photo-crosslinking method, differential interactions of proteins with mutant and wild type nascent chains translated in vitro have revealed a previously unappreciated mechanism for preemptive quality control. The system involves proteins that lead to the degradation of the mRNA coding for the mutant protein, thereby reducing the production of protein bound to misfold. This mechanism prevents the accumulation of potentially cytotoxic misfoided proteins without spending energy for futile translation and subsequent ATP dependent proteolysis by the proteasome. We are now requesting continued support of the MERIT award to extend the analyses successfully applied to AF508 in Aim 1 and 2 to additional CF-causing mutations and to define and characterize the mechanisms responsible for preemptive quality control system discovered during execution of Aim 3. We thank the institute for selecting our study for MERIT support that allowed pursuit of the long term discovery effort of Aim 3 that has now revealed novel and unexpected biology. Such a path would not have been feasible under the time constraints of a R01. RELEVANCE (See instructions): Most cases of cystic fibrosis, a common fatal genetic disease, are caused by mutations that interfere with the assembly of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The studies proposed will elucidate the details of how the disease-causing mutations interfere with this process. Understanding the assembly process, and, thus, the detailed molecular pathology, will provide important information for developing targeted therapeutics for cystic fibrosis.