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）是第一个涉及内质网相关降解（ERAD）的底物的哺乳动物蛋白，它已被用作多面体膜蛋白折叠的模型。由于其复杂的折叠和结构域组装要求，大多数WT CFTR和~100%的普通折叠突变体，？F508 CFTR，被泛素-蛋白酶体系统降解。我们发现了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 Hsp27选择性地降解？F508雌性生殖道。这种作用可以通过hsp27介导的CFTR与小的泛素样修饰剂SUMO的偶联来解释。重要的是，敲除Hsp27或E1 SUMO转移酶可使CFTR表达增加2-3倍。CFTR三个一致sumo化位点的突变降低了WT和？F508 CFTR表达量~80%，同时也消除了Hsp27和SUMO E2促进？F508 CFTR降解。这些发现导致了这样的假设，即Hsp27介导的WT CFTR的sumoylation在天然结构的组装过程中保持了域溶解度，并且Hsp27和SUMO未能有效地从？F508 CFTR以降解为目标。我们将使用诱变、生化和功能分析来确定影响CFTR生物发生的sumo化位点。Hsp27结合和sumo化与WT和？F508 NBD1和全长CFTR将使用多种方法进行评估：生物物理分析将包括固有色氨酸荧光和圆二色性，一种新的NBD1溶解度酶分析，有限蛋白水解将报告NBD1和全长蛋白的紧密性，作为Hsp27结合和SUMO修饰的功能。渐进的c端CFTR截断将探索CFTR结构域对Hsp27结合和SUMO化的依赖性，而CFTR翻译过程中与Hsp27和SUMO的交联将评估这些过程的早期步骤。这些方法也将阐明CFTR summoylation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，其在CFTR sumoylation中的作用将被评估。对其他CFTR折叠突变体和其他蛋白质折叠疾病构象突变体的研究将检验Hsp27- SUMO介导的稳定/降解的普遍性。该项目首次提供了sHsp参与CFTR生物发生的证据，以及SUMO与sHsp客户蛋白结合的证据。本研究将阐明该通路在WT CFTR折叠和突变型CFTR降解中的分子机制和意义，并为评估该系统在其他蛋白质构象疾病中的意义提供一种途径。公共卫生相关性：本项目将评估小热休克蛋白（sHsp）介导的sumoylation启动常见CFTR突变体F508降解的假设。作为次要假设，将确定瞬时sHsp结合和sumoylation在野生型CFTR折叠和组装中的作用。这个新颖的概念，即sHsp催化SUMO转移到它们的底物，将被评估为sHsp伴侣功能的一般机制。

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.