Wong Swee-Hee, Wang Wen-Hung, Chen Pin-Hua, Li Shuan-Yow, Yang Jiann-Jou
Department of BioMedical Sciences, Chung Shan Medical University, Taichung, Taiwan;; Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.
Department of Otolaryngology, Cathay General Hospital, Taipei, Taiwan.
Int J Med Sci. 2017 Feb 23;14(3):246-256. doi: 10.7150/ijms.17785. eCollection 2017.
In a previous study, we identified a novel missense mutation, p.W77S, in the gene encoding connexin30.2/connexin31.3 (CX30.2/CX31.3) from patients with hearing loss. The functional alteration of CX30.2/CX31.3 caused by the p.W77S mutant of gene, however, remains unclear. In the current study, our result indicated that the p.W77 is localized at the second membrane-spanning segments (TM2) and near border of the E1 domain of the CX30.2/CX31.3 protein and highly conserved (Conseq score = 8~9) in all species. The p.W77S missense mutation proteins in the intracellular distribution are different CX30.2/CX31.3WT and an accumulation of the mutant protein in the endoplasmic reticulum (ER) of the HeLa cell. Furthermore, co-expression of WT and p.W77S mutant chimerae proteins showed that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the WT proteins' expression in the cell membranes. In addition, we found that CX30.2/CX31.3W77S missense mutant proteins were degraded by lysosomes and proteosomes in the transfected HeLa cell. Based on these findings, we suggest that p.W77S mutant has a dominant negative effect on the formation and function of the gap junction. These results give a novel molecular elucidation for the mutation of in the development of hearing loss.
在之前的一项研究中，我们在患有听力损失的患者中，于编码连接蛋白30.2/连接蛋白31.3（CX30.2/CX31.3）的基因中鉴定出一种新的错义突变p.W77S。然而，该基因突变的p.W77S突变体所导致的CX30.2/CX31.3功能改变仍不清楚。在当前研究中，我们的结果表明，p.W77位于CX30.2/CX31.3蛋白的第二个跨膜片段（TM2）以及E1结构域边界附近，并且在所有物种中高度保守（保守性评分=8至9）。细胞内分布的p.W77S错义突变蛋白与CX30.2/CX31.3野生型不同，并且突变蛋白在HeLa细胞的内质网（ER）中积累。此外，野生型和p.W77S突变体嵌合蛋白的共表达表明，异源连接子在细胞质中积累，从而损害了野生型蛋白在细胞膜上的表达。另外，我们发现CX30.2/CX31.3W77S错义突变蛋白在转染的HeLa细胞中被溶酶体和蛋白酶体降解。基于这些发现，我们认为p.W77S突变体对缝隙连接的形成和功能具有显性负效应。这些结果为听力损失发生过程中的基因突变提供了一种新的分子解释。
