Loss of function and impaired degradation of a cataract-associated mutant connexin50
Loss of function and impaired degradation of a cataract-associated mutant connexin50
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DOI：
10.1078/0171-9335-00316
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发表时间：
2003-05-01
影响因子：
6.6
通讯作者：
Beyer, EC
中科院分区：
文献类型：
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作者：
Berthoud, VM;Minogue, PJ;Beyer, EC
突变的人类连接蛋白50(HCx50)，hCx50P88S，已被认为与白内障有关，作为常染色体显性遗传性状。在转基因细胞中检测野生型和突变型hCx50的功能、生化和细胞行为。HCx50P88S不能单独诱导缝隙连接电流，与野生型(Wt)hCx50共表达时，hCx50P88S可消除缝隙连接电流。转染hCx50P88S的细胞除在贴壁膜染色外，还可见胞浆内CX50免疫反应阳性物质的聚集；这些聚集与内质网、高尔基体、溶酶体、内小体或波形蛋白细丝的标记没有显著的共定位。免疫电子显微镜研究hCx50P88S定位于靠近内质网的细胞膜堆叠。相反，用蛋白酶体抑制剂处理wt hCx50转基因细胞可诱导侵袭体样聚集。Brefeldin A不能阻止瞬时转染细胞中hCx50P88S的形成，这表明它们是在CX50通过高尔基体转运到质膜之前形成的。用放线菌酮处理HeLa-hCx50P88S细胞，发现存在一个非常稳定的hCx50P88S池。综上所述，这些结果表明，氨基酸残基88位的P到S突变导致了缺陷，导致hCx50P88S在不同于侵袭体的细胞结构中降解减少和随后积累。
A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when coexpressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations, were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.