DEVELOPMENT OF MEMBRANE PROTEIN STRUCTURE

项目总监/首席调查员(最后第一，中)：S，L，P。 项目总结(见说明)： 被选为优异奖的R01奖助金的竞争性续签的原始申请 提出了三项救济措施，以解决与CFTRI机制有关的三个基本问题 形成其功能和天然结构，以及这一过程是如何被致病突变改变的。多么 AF508是否干扰NBD1折叠？AF508是否显著修改了 将NBD1与其他域合并？与质量控制蛋白的哪些相互作用是关键的？在.期间 在最初的四年半的功绩支持中，我们已经回答了前两个问题 道路。这些结果表明，CFTR折叠是一个分层的过程，并为 纠正AF508突变体折叠的现有化合物的功效“天花板”。他们还建议 以机理为基础的方法来发现与现有化合物协同工作的新化合物 校正剂或绕过“天花板”的新型化合物。这些方法已经被采用。 通过一些药物发现的努力。最后，使用一种强大的特定光交联法， 蛋白质与体外翻译的突变型和野生型新生链的不同相互作用 一种以前未被认可的先发制人的质量控制机制。该系统涉及到的蛋白质 导致编码突变蛋白的mRNA的降解，从而减少 蛋白质会发生错误折叠。这一机制防止了潜在的细胞毒性错误堆积。 蛋白质没有花费能量进行无用的翻译和随后的ATP依赖的蛋白质分解 蛋白酶体。我们现在请求继续支持优秀奖，以延长分析 成功应用于目标1和目标2中的AF508，用于其他导致CF的突变，并定义和 描述在执行过程中发现的负责先发制人的质量控制体系的机制 目标3.我们感谢研究所选择我们的研究作为功绩支持，使我们能够长期追求 目标3的术语发现工作，现在揭示了新的和意想不到的生物学。这样的道路不会 在R01的时间限制下是可行的。 相关性(请参阅说明)： 囊性纤维化是一种常见的致命遗传病，大多数病例是由干扰 囊性纤维化跨膜电导调节蛋白的组装。这项研究建议 将阐明致病突变如何干扰这一过程的细节。了解 组装过程，以及详细的分子病理学，将为 开发囊性纤维化的靶向治疗药物。

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.