Chaperone Actions in CFTR Biogenesis

描述(申请人提供)：囊性纤维化跨膜电导调节器(CFTR)是第一个被认为是内质网相关降解(ERAD)底物的哺乳动物蛋白，它已经成为折叠多角体膜蛋白的模型。由于其复杂的折叠和结构域组装要求，大部分WT cftr和~100%的常见折叠突变体？F508cftr被泛素-蛋白酶体系统降解。我们发现了CFTR和小分子热休克蛋白(SHsps)之间的一种新的相互作用，并表明主要的呼吸道细胞SHSP，Hsp27，选择性地降解F508 CFTR。这一作用是由Hsp27介导的CFTR与小泛素样修饰物SUMO的结合来解释的。重要的是，Hsp27或E1相扑转移酶的敲除使CFTR的表达增加了2-3倍。CFTR3个共同的总甲基化位点突变使WT和F508CFTR的表达降低约80%，同时也使Hsp27和相扑E2不能促进F508CFTR的降解。这些发现导致了这样的假设，即Hsp27介导的WT CFTR在天然结构组装过程中保持了结构域的溶解性，并且未能有效地从F508 CFTR中去除Hsp27和SUMO以其降解为目标。我们将使用突变、生化和功能分析来确定影响CFTR生物发生的苏莫化位点。Hsp27的结合和总甲基化与WT和F508 NBD1的构象和全长CFTR的关系将用多种方法来评估：生物物理分析将包括固有的色氨酸荧光和圆二色谱，一种新的NBD1溶解性的酶促分析，而有限蛋白质分解将报告NBD1和全长蛋白质的致密性是Hsp27结合和SUMO修饰的函数。渐进性的C端CFTR截断将探索Hsp27结合和SUM化的CFTR域依赖性，而在CFTR翻译过程中与Hsp27和SUMO的交叉连接将评估这些过程的早期步骤。这些方法还将阐明CFTR总甲基化与核心伴侣蛋白相互作用和已知CFTR降解途径的关系。大多数sHsps中都存在一个相扑相互作用基序(SIM)，我们将评估它在CFTR相思酰化中的作用。对其他CFTR折叠突变体和其他蛋白质折叠疾病的构象突变的研究将检验Hsp27-SUMO介导的稳定/降解的普遍性。该项目提供了SHSP参与CFTR生物发生的第一个证据，以及相扑与SHSP客户蛋白的结合。这项研究将阐明该途径在WT CFTR折叠和突变CFTR降解中的分子机制和意义，并将为评估该系统在其他蛋白质构象疾病中的意义提供一个门户。公共卫生相关性：该项目将评估一种假说，即小分子热休克蛋白(SHSP)介导的苏莫化启动常见的CFTR突变体F508的降解。作为第二个假设，将确定瞬时SHSP结合和总甲基化在野生型CFTR折叠和组装中的作用。这一新的概念，即sHSPs催化相扑转移到它们的底物上，将作为SHSP伴侣功能的一般机制进行评估。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) was the first mammalian protein implicated as a substrate for endoplasmic reticulum associated degradation (ERAD), and it has served as a model for the folding of polytopic membrane proteins. Due to its complex folding and domain assembly requirements, the majority of WT CFTR and ~100% of the common folding mutant, ?F508 CFTR, are degraded by the ubiquitin-proteasome system. We identified a novel interaction between CFTR and small heat shock proteins (sHsps), and showed that the predominant airway cell sHsp, Hsp27, selectively degrades ?F508 CFTR. This action was explained by Hsp27-mediated conjugation of CFTR with the small ubiquitin-like modifier, SUMO. Importantly, knockdown of Hsp27 or the E1 SUMO transfer enzyme increased CFTR expression 2-3 fold. Mutation of CFTR's three consensus sumoylation sites reduced WT and ?F508 CFTR expression ~80%, and also eliminated the ability of Hsp27 and the SUMO E2 to promote ?F508 CFTR degradation. These findings led to the hypothesis that Hsp27-mediated sumoylation of WT CFTR maintains domain solubility during assembly of the native structure, and that the failure to efficiently remove Hsp27 and SUMO from ?F508 CFTR targets its degradation. We will use mutagenesis, biochemical and functional assays to identify the sumoylation sites that influence the biogenesis of CFTR. The relation of Hsp27 binding and sumoylation to the conformations of WT and ?F508 NBD1 and full-length CFTR will be assessed using multiple methods: biophysical assays will include inherent tryptophan fluorescence and circular dichroism, a new enzymatic assay of NBD1 solubility, and limited proteolysis will report on compactness of NBD1 and full-length protein as a function of Hsp27 binding and SUMO modification. Progressive C-terminal CFTR truncations will explore the CFTR domain-dependence of Hsp27 binding and sumoylation, and cross-linking to Hsp27 and SUMO during CFTR translation will assess early steps in these processes. These approaches will also illuminate the relation of CFTR sumoylation to core chaperone protein interactions and to known CFTR degradation pathways. A SUMO interacting motif (SIM) is present in most sHsps, and its role in CFTR sumoylation will be evaluated. Studies of other CFTR folding mutants and of conformational mutants in other protein folding diseases will examine the generality of Hsp27- SUMO mediated stabilization/degradation. This project has provided the first evidence of sHsp involvement in CFTR biogenesis, and of SUMO conjugation to sHsp client proteins. The proposed research will clarify the molecular mechanism and the significance of this pathway in WT CFTR folding and mutant CFTR degradation, and it will provide a gateway to evaluate the significance of this system in other diseases of protein conformation. PUBLIC HEALTH RELEVANCE: This project will evaluate the hypothesis that small heat shock protein (sHsp)-mediated sumoylation initiates the degradation of the common CFTR mutant, ?F508. As a secondary hypothesis, the role of transient sHsp binding and sumoylation in the folding and assembly of wild type CFTR will be determined. This novel concept, that sHsps catalyze the transfer of SUMO to their substrates, will be evaluated as a general mechanism of sHsp chaperone function.