| Budget Amount *help |
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2002: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Research Abstract |
A major hallmark of the polyglutamine (pQ) diseases is the formation of pQ inclusions. Recently, misfolding has come to be considered one of the primary factors for pQ protein aggregation, although, the nature of misfolding and the relationship between misfolding and ubiquitination of the expanded pQ protein is not yet known. By using ataxin-3, the defective gene product of SCA3/MJD, we demonstrated that the cellular toxicity of a pQ protein is directly proportional to the length of the glutamine repeats and inversely dependent on the size of the corresponding protein. The pQ expansion and truncation enhance the reactivity of 1C2 antibody (specific to expanded pQ), chaperone association and ubiquitination, suggesting that pQ expansion and protein truncation enhance the protein misfolding.
The protein misfolding induced by pQ expansion was further investigated with our molecular model system using mutant myoglobin which is inserted different size of pQ. Polyglutamine stretches form intra
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molecular β-sheets when pQ expanded and they form intermolecular β-sheets and amyloid fibrils after certain incubation time. The surface of the mutant myoglobin with expanded pQ was partially unfolded and destabilized. We also investigated the early phase of fibrillization of the mutant myoglobin in which 35 or 50 glutamine repeats are inserted. Small-angle X-ray scattering and electron microscopic studies revealed that the expansion of pQ to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. We found that β-sheets of pQ in quasi-aggregate were exposed on the surface and were not regularly oriented, as opposed to those in amyloid fibril. X-ray diffraction analysis indicated that the expansion of glutamine repeat did not show substantial effects on the β-sheet structure of pQ in fibril. Since the expansion of glutanine repeat in certain proteins is responsible for pQ diseases, the present study suggests that the condensed and non-oriented β-sheets exposed on the surface of quasi-aggregate, rather than the regularly aligned β-sheets in fibrils, are closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pQ diseases. Thus, the quasi-aggregate form also should be targeted to the therapeutic strategy for pQ diseases.
Base on those structural change, we searched for compounds to stabilize the molecule with expanded polyglutamine and found some effective compounds. Less
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