Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach.
Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach.
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DOI：
10.1074/jbc.m611049200
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发表时间：
2007-03
期刊：
影响因子：
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通讯作者：
Michael Wolde;Abigail Fellows;Jie Cheng;Aleksandr Kivenson;B. Coutermarsh;L. Talebian;K. Karlson;A. Piserchio;D. Mierke;B. Stanton;W. Guggino;D. Madden
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文献类型：
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作者：
Michael Wolde;Abigail Fellows;Jie Cheng;Aleksandr Kivenson;B. Coutermarsh;L. Talebian;K. Karlson;A. Piserchio;D. Mierke;B. Stanton;W. Guggino;D. Madden
PDZ结构域是普遍存在的肽结合模块，在各种细胞内运输和定位过程中介导蛋白质-蛋白质相互作用。这些包括调节囊性纤维化跨膜传导调节剂（CFTR）的膜运输和内吞循环的途径，CFTR是囊性纤维化患者中发生突变的一种上皮氯离子通道。相应地，许多PDZ蛋白现在已经被确定直接或间接地与CFTR的C端相互作用。其中之一是CAL，其在异源细胞中的过表达以剂量依赖的方式指导WT-CFTR的溶酶体降解，并减少在细胞表面发现的CFTR的数量。在这里，我们发现靶向内源性CAL的RNA干扰特异性地增加了疾病相关的DeltaF508-CFTR突变体的细胞表面表达，从而增强了极化的人患者支气管上皮细胞系的上皮氯电流。我们用纯化的组分在体外重建了CAL-CFTR相互作用，首次证明了这种结合是直接的，并使我们能够从生物化学和生物物理上表征其组分。为了验证结合位点的抑制也可以逆转CAL介导的CFTR抑制的假设，构建了CAL.CFTR复合物的三维同源模型，并使用该模型生成了一个CAL突变体，其结合口袋折叠正确，但失去了结合CFTR的能力。虽然产生的水平与野生型蛋白相同，但突变体不影响CFTR的表达水平。综上所述，我们的数据确立了CAL作为纠正囊性纤维化中成熟后转运缺陷的候选治疗靶点。
PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.