| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2011: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2009: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Research Abstract |
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
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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
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