| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
To study the function of protein p40, which is specifically decreased in tuberous sclerosis complex, relations between p40 and tuberin, hamartin were investigated using immunoprecipitation and western blotting. HeLa cells cultured on 6 cm dishes were lysed using RIPA buffer and span for 10 minutes at 10,000g. The lysates were mixed with anti-p40, anti-tuberin or anti-hamartin antibodies. To the mixture, protein G Sepharose 4B was added to precipitate. These antrigen antibody complex precipitates were subjected to SDS-PAGE. Co-precipitated molecules were detected by westernblotting using the antibodies described above.
As a result, hamartin bound to p40, but it is not clear whether tuberin bound to p40. To verify these results, p40 gene was knocked down and the effect of suppression of p40 on the expression of tuberin and hamartin was studied. Knocking down of p40 using p40-specific siRNA increased the amount of tuberin, but did not increase the phosphorylated tuberin. Meanwhile, overexpression of p40 induced apoptosis in tuberous sclerosis cells, although overexpression of p40 did not induce apoptosis in normal cells.
Considering these results, we have a following hypothesis. P40 binds to hamartin. So decrease of p40 inhibits complex formation of p40 and hamartin. This decrease of p40-hamartin complex results in increase of free hamartin. Free hamartin binds to tuberin and forms tuberin-hamartin complex. Increased tuberin-hamartin complex suppresses Rheb activity. On the other hand, transfection of p40 in tuberous sclerosis cells without hamartin increases free p40 prominently and induces apoptotic cell death. P40 is essential for cell survival, but only small amount of p40 is sufficient for cell survival. It is likely that too much of p40 may be toxic.
Although the mechanism is still unknown, p40 may be useful as TSC tumor suppression tool because transfection of p40 with tuberous sclerosis cells increased apoptosis specifically.
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