BIOSYNTHETIC ARREST OF CFTR

该项目的研究人员建议阐明 参与野生型CFTR的新型生物合成过程及其 在delta F508和其他疾病相关变体的情况下失败。 迄今为止所做的研究表明，这些机制可能会 错综复杂。 我们已经了解到，新生CFTR 在内质网（ER）两侧有分子伴侣 膜，并在细胞质侧被泛素识别， 蛋白酶体途径 我们假设CFTR的协调组装 多个胞质结构域与12个膜- 跨越序列需要几个伴侣蛋白， 培育一个天然的整体三级结构或失败，并导致分子 降解途径。 伴侣的这种双重作用可以提供 有效的动力学机制来处理无法实现 成熟的折叠状态，尽管重复的伴侣结合轮。 的 涉及蛋白水解途径的比例可以特别 对于像CFTR这样具有复杂结构域的蛋白质， 复杂的调节功能。 我们将继续这个假设， 四个具体目标。第一个并不深刻，只是为了收集 直接证据表明，观察到野生型CFTR的低效成熟 实际上发生在体内相关的上皮组织中。 第二，进一步剖析新生的 CFTR与分子伴侣，泛素-蛋白酶体系统和其他 降解途径 我们将确定泛素化的位点， CFTR，并确定蛋白质的C-末端尾部在 决定成熟和退化之间的平衡。 的 初步证据表明，R-结构域可能是特别重要的 将新生CFTR靶向于泛素化和蛋白水解， 进一步探索。 在目标3中，我们将利用酵母突变体， 分泌途径，在泛素-蛋白酶体途径和分子 伴侣来剖析ER处CFTR处理中的步骤。在目标4中， 将继续我们对疾病相关突变的系统评价， 以确定哪些会导致错误处理。刚刚完成 分析了30个这样的突变在细胞质环，我们现在将转向 与跨膜序列有关。 这一目标的第二部分是 试图确定这些突变对C1通道的影响， 通过将来自表达它们的细胞的微粒体与平面脂质融合来发挥功能 双层。

The investigators of this project propose to elucidate the mechanisms involved in the novel biosynthetic processing of wild-type CFTR and its failure in the case of delta F508 and other disease-associated variants. Studies done thus far have indicated that these mechanisms are likely to be intricate and complex. We have learned that nascent CFTR interacts with molecular chaperones on both sides of the endoplasmic reticulum (ER) membrane and is recognized on the cytoplasmic side by the ubiquitin- proteasome pathway. We postulate that the coordinated assembly of CFTR's multiple cytoplasmic domains with the integration of twelve membrane- spanning sequences requires several chaperones which either succeed in fostering a native global tertiary structure or fail and lead the molecule to degradation pathways(s). This dual role of chaperones may provide an efficient kinetic mechanism to dispose of molecules unable to achieve a mature folded state despite repeated rounds of chaperone binding. The proportion which are directed to a proteolytic pathway may be especially high for proteins like CFTR with an elaborate domain structure necessary to its complex regulatory function. We shall pursue this hypothesis by four specific aims. The first is not profound and aims simply to collect direct evidence that the inefficient maturation of wild-type CFTR observed in cultured cells actually occurs in vivo in relevant epithelial tissues. The second is to further dissect the network of interactions of nascent CFTR with chaperones, the ubiquitin-proteasome system and other degradation pathways. We shall identify the sites of ubiquitination on CFTR and determine the exact role of the C-terminal tail of the protein in determining the balance between maturation and degradation. The preliminary evidence that the R-domain may be especially important in the targeting of nascent CFTR for ubiquitination and proteolysis will be explored further. In Aim 3 we shall utilize yeast mutants in the secretory pathway, in the ubiquitin-proteasome pathway and in molecular chaperones to dissect the steps in CFTR processing at the ER. In Aim 4, we shall continue our systematic evaluation of disease-associated mutations to determine which ones cause misprocessing. Having just completed analysis of 30 such mutations in the cytoplasmic loops, we shall now turn to the membrane-spanning sequences. The second part of this aim is to attempt to determine the influence of these mutations on in C1- channel function by fusing microsomes from cells expressing them with planar lipid bilayers.