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

TACSTD2基因调节上皮紧密连接的机制的阐明

基本信息

批准号：
21592238
负责人：
KAWASAKI Satoshi
金额：
$ 2.83万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

项目摘要

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

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