Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 2013 Jul 12;288(28):20427-34. doi: 10.1074/jbc.M113.452847. Epub 2013 May 17.
The mechanisms by which mutant connexins lead to disease are diverse, including those of connexin50 (CX50) encoded by the GJA8 gene. We investigated the cellular and functional behavior of CX50fs, a mutant CX50 that has a frameshift after amino acid 255 and causes recessive congenital cataracts. Cellular levels of CX50fs were much lower than those of wild type CX50 in stably transfected HeLa cells. Whereas CX50 localized at distinct gap junction plaques and supported extensive intercellular transfer of Neurobiotin, CX50fs gap junctions were rare, and their support of Neurobiotin transfer was reduced by >90%. After inhibition of new protein synthesis with cycloheximide, CX50fs disappeared much more rapidly than CX50, suggesting increased degradation of the mutant. Treatment of cells with epoxomicin (a proteasomal inhibitor) led to a dramatic increase in CX50fs levels and in the abundance of gap junctions. Epoxomicin treatment also rescued intercellular transfer of Neurobiotin to levels similar to those in cells expressing the wild type protein. Treatment with eeyarestatin I (an inhibitor of p97-dependent protein degradation) resulted in many abundant slowly migrating CX50 and CX50fs bands consistent with polyubiquitination of the proteins. These results demonstrate that the CX50fs mutant is rapidly degraded by endoplasmic reticulum-associated degradation in mammalian cells. This accelerated degradation reduces the abundance of gap junctions and the extent of intercellular communication, potentially explaining the pathogenesis of cataracts linked to this mutant. The efficacy of epoxomicin in restoring function suggests that protease inhibition might have therapeutic value for this and other diseases caused by mutants with similar defects.
突变连接蛋白导致疾病的机制多种多样，包括 GJA8 基因编码的连接蛋白 50（connexin50，CX50）。我们研究了 CX50fs 的细胞和功能行为，这是一种突变型 CX50，其在氨基酸 255 后发生移码，导致隐性先天性白内障。在稳定转染的 HeLa 细胞中，CX50fs 的细胞内水平明显低于野生型 CX50。虽然 CX50 定位于独特的缝隙连接斑上，并支持 Neurobiotin 的广泛细胞间转移，但 CX50fs 缝隙连接很少，其支持 Neurobiotin 转移的能力降低了>90%。在用环己酰亚胺（一种蛋白质合成抑制剂）抑制新蛋白质合成后，CX50fs 的消失速度比 CX50 快得多，这表明突变体的降解增加。用环氧体素（一种蛋白酶体抑制剂）处理细胞会导致 CX50fs 水平和缝隙连接丰度显著增加。环氧体素处理还挽救了 Neurobiotin 的细胞间转移，使其达到表达野生型蛋白的细胞的水平。用 eeyarestatin I（一种依赖 p97 的蛋白质降解抑制剂）处理会导致大量丰富的缓慢迁移的 CX50 和 CX50fs 带，这与蛋白质的多泛素化一致。这些结果表明，在哺乳动物细胞中，CX50fs 突变体通过内质网相关降解迅速降解。这种加速降解降低了缝隙连接的丰度和细胞间通讯的程度，可能解释了与该突变体相关的白内障的发病机制。环氧体素恢复功能的功效表明，蛋白酶抑制剂可能对这种突变体和其他具有类似缺陷的疾病具有治疗价值。
