Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism.
Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism.
复制标题
Rab27a 负向调节结肠上皮中的 CFTR 氯离子通道功能：效应蛋白参与调节机制。
DOI：
10.1016/j.bbrc.2006.05.102
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发表时间：
2006
影响因子：
3.1
通讯作者：
Kaur,Simarna
中科院分区：
文献类型：
--
作者：
Saxena,SunilK;Kaur,Simarna
囊性纤维化是一种常染色体隐性遗传病，由CFTR的生物合成或功能破坏引起。CFTR的调控机制包括通道转运到质膜和蛋白-蛋白相互作用。Rab蛋白是参与囊泡运输、对接和融合的小gtp酶。结直肠上皮细胞HT-29细胞天然表达CFTR，并随着CFTR介导电流的增加而对cAMP产生反应。dpc抑制电流可以被cftr特异性SiRNA完全消除。过表达Rab27a抑制HT-29细胞中cftr介导的电流，而同种异构体特异性SiRNA和Rab27a抗体则刺激cftr介导的电流。CFTR活性被Rab27a (Q78L) （Rab27a的组成活性gtp结合形式）和Rab27a (T23N)（模仿gdp结合形式的组成阴性形式）抑制。Rab27a介导的效应可被Rab27a结合蛋白、突触蛋白样蛋白（SLP-5）和Munc13-4辅助蛋白（一种推测的胞外分泌启动因子）逆转。SLP逆转Rab27a的作用仅限于C2A/C2B结构域，而SHD基序的抑制作用较小。cftr介导的电流不受Rab3的影响，尽管SLP-5似乎与Rab3弱结合。免疫沉淀实验表明Rab27a和CFTR之间存在蛋白-蛋白相互作用。Rab27a似乎通过将CFTR捕获在细胞内区室中来损害CFTR在细胞表面的外观。另一方面，Munc13-4和SLP-5限制了Rab27a对CFTR的可用性，从而使其对通道功能的影响最小化。这些观察结果有力地证明了Rab27a通过Munc13-4和SLP-5效应蛋白的蛋白-蛋白相互作用参与CFTR通道调控，因此可能成为囊性纤维化治疗的潜在靶点。
Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein–protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein–protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein–protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.