BIOSYNTHETIC ARREST OF CFTR

该项目的研究人员建议阐明这些机制。 参与野生型CFTR新的生物合成工艺及其 在Delta F508和其他与疾病相关的变种的情况下失败。 到目前为止所做的研究表明，这些机制可能会 要错综复杂。我们已经了解到，新生的CFTR相互作用 内质网两侧均有分子伴侣 并在细胞质一侧被泛素识别- 蛋白酶体途径。我们假设CFTR的协调集会 多个细胞质结构域，整合了12个膜- 生成序列需要几个伴侣，这些伴侣要么成功 培育天然的全球三级结构或失败并领导分子 到退化途径(S)。监护人的这种双重角色可以提供一种 高效的动力学机制来处理无法实现的分子 成熟的折叠状态，尽管有反复的伴侣结合。这个 与蛋白分解途径有关的比例可能特别 对于像CFTR这样具有复杂结构的蛋白质来说很高 其复杂的监管职能。我们将通过以下方式进一步研究这一假说 四个具体目标。第一个并不深奥，目的只是为了收集 观察到野生型cftr低效成熟的直接证据 在培养的细胞中，实际上发生在体内的相关上皮组织中。 二是进一步剖析新生生物相互作用的网络 CFTR与伴侣、泛素-蛋白酶体系统及其他 退化途径。我们将确定泛素化的位置在 Cftr，并确定该蛋白的C-末端尾部在 决定成熟和退化之间的平衡。这个 初步证据表明，R-结构域可能在 新生的cftr泛素化和蛋白降解的靶点将是 进一步探索。在目标3中，我们将利用酵母突变株在 泛素-蛋白酶体途径和分子中的分泌途径 在急诊室仔细分析CFTR处理步骤的监护人。在目标4中，我们 将继续我们对疾病相关突变的系统评估 以确定哪些原因会导致误处理。刚刚完工 分析了细胞质环中的30个这样的突变，现在我们将转向 到跨膜序列。这一目标的第二部分是 试图确定这些突变对In C_1通道的影响 将表达微粒的细胞中的微粒与平面脂类融合的功能 双层的。

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.