The elucidation of the mechanism regulating the epithelial tight-junction by the TACSTD2 gene

在本研究中，我们试图研究TACSTD 2基因功能缺失突变的机制。凝胶状滴状营养不良（GDLD）的责任基因。参与GDLD的发病机制，以及开发针对该疾病的治疗。TACSTD 2基因编码具有1个跨膜结构域和短胞内区域的I型膜蛋白。我们发现，TACSTD 2蛋白结合的claudin 1和7蛋白，这是上皮细胞紧密连接的主要组成部分，使用免疫沉淀法。我们还发现，如果我们敲低TACSTD 2基因，紧密连接相关蛋白（包括claudin 1和7蛋白）的亚细胞定位会随着上皮屏障功能的降低而显著改变。我们还发现，与正常对照组相比，GDLD患者角膜上皮中claudin 1、4和7蛋白的表达水平显著降低，而其mRNA水平无变化。 ...更多信息 角膜上皮我们将claudin 1、4或7基因转导到HeLa细胞中，有或没有TACSTD 2基因，并且发现当与TACSTD 2基因共转导时，claudin 1和7的表达水平显著增加。接下来，我们用MG-132（一种强蛋白酶体抑制剂）处理了用claudin 1或7基因转导的HeLa细胞。根据上述结果，我们发现MG-132处理显著增加了密蛋白1或7蛋白的表达水平。这些数据表明，TACSTD 2蛋白对claudin 1和7的蛋白降解具有保护作用，可能是通过泛素-蛋白酶体蛋白降解途径。接下来，我们使用免疫沉淀测定研究了密蛋白1和7蛋白的泛素化。然而，我们意外地发现，claudin 1和7蛋白根本没有泛素化。这与来自GDLD患者的永生化角膜上皮细胞的上皮屏障功能没有通过MG-132的治疗得到改善的结果非常一致。少

In the present study, we sought to investigate the mechanism by which the loss of function mutation of the TACSTD2 gene. the responsible gene for gelatinous drop-like dystrophy(GDLD). is involved in the pathogenesis of the GDLD, as well as to develop a therapy for this disease. The TACSTD2 gene encodes a type I membrane protein with 1 transmembrane domain and a short intracellular region. We found that the TACSTD2 protein binds to the claudin 1 and 7 proteins, which are major components of epithelial-tight-junction-using immunoprecipitation assay. We also found that if we knocked down the TACSTD2 gene, the subcellular localization of the tight-junction-related proteins, including the claudin 1 and 7 proteins, was significantly altered with the decrease in epithelial barrier function. We also found that the expression level of the claudin 1, 4, and 7 proteins in the corneal epithelium of GDLD patients was significantly decreased, while their mRNA level was unchanged compared to normal c … More orneal epithelium. We transduced the claudin 1, 4 or 7 gene to HeLa cells, with or without the TACSTD2 gene, and found that the expression level of claudin 1 and 7 was significantly increased when they were co-transduced with the TACSTD2 gene. We next treated the HeLa cells, which were transduced with the claudin 1 or 7 gene, with MG-132, a strong proteasome inhibitor. In accordance with the above results, we found that the MG-132 treatment significantly increased the expression level of the claudin 1 or 7 proteins. These data suggest that the TACSTD2 protein has a protective role against the protein degradation of claudin 1 and 7, possibly through the ubiquitin-proteasome protein degradation pathway. We next investigated the ubiquitination of the claudin 1 and 7 proteins using immunoprecipitation assay. However, we unexpectedly found that the claudin 1 and 7 proteins were not at all ubiquitinated. This was in good agreement with the results that the epithelial barrier function of immortalized corneal epithelial cells derived from a GDLD patient was not improved by the treatment of MG-132. Less