Chaperone Actions in CFTR Biogenesis

描述（由申请人提供）：囊性纤维化跨膜传导调节因子（CFTR）是第一个涉及作为内质网相关降解（ERAD）底物的哺乳动物蛋白，并且其已用作多位膜蛋白折叠的模型。由于其复杂的折叠和结构域组装要求，大多数WT CFTR和~100%的常见折叠突变体，？F508 CFTR被泛素-蛋白酶体系统降解。我们确定了CFTR和小热休克蛋白（sHsps）之间的一种新的相互作用，并表明，主要的气道细胞sHsp，Hsp 27，选择性降解？F508 CFTR。这一作用被解释为热休克蛋白27介导的共轭CFTR与小泛素样修饰，SUMO。重要的是，Hsp 27或E1 SUMO转移酶的敲低使CFTR表达增加2-3倍。突变CFTR的三个共识sumoylation网站减少WT和？F508 CFTR表达~ 80%，并且还消除了Hsp 27和SUMO E2促进？F508 CFTR降解。这些发现导致了这样的假设，即Hsp 27介导的SUMO化WT CFTR在组装天然结构期间保持结构域的溶解性，并且未能有效地去除Hsp 27和SUMO？F508 CFTR针对其降解。我们将使用诱变，生化和功能测定，以确定sumoylation网站，影响CFTR的生物发生。热休克蛋白27的结合和sumo化的WT和？F508 NBD 1和全长CFTR将使用多种方法进行评估：生物物理测定将包括固有色氨酸荧光和圆二色性，NBD 1溶解度的新酶促测定，以及有限的蛋白水解将报告NBD 1和全长蛋白质的致密性作为Hsp 27结合和SUMO修饰的函数。渐进的C末端CFTR截短将探索Hsp 27结合和SUMO化的CFTR结构域依赖性，而CFTR翻译期间与Hsp 27和SUMO的交联将评估这些过程的早期步骤。这些方法还将阐明CFTR类小泛素化与核心伴侣蛋白相互作用和已知CFTR降解途径的关系。SUMO相互作用基序（SIM）存在于大多数sHsps中，并且将评估其在CFTR类小泛素化中的作用。对其他CFTR折叠突变体和其他蛋白质折叠疾病中的构象突变体的研究将检查Hsp 27- SUMO介导的稳定化/降解的一般性。该项目提供了sHsp参与CFTR生物发生以及SUMO与sHsp客户蛋白缀合的第一个证据。该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途径。公共卫生相关性：该项目将评估假设小热休克蛋白（sHsp）介导的类小泛素化启动常见CFTR突变体的降解，？F508作为次要假设，将确定瞬时sHsp结合和类小泛素化在野生型CFTR的折叠和组装中的作用。这一新的概念，即sHsps催化SUMO转移到其底物，将作为sHsp分子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.