| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
DNA-dependent protein kinase (DNA-PK) phosphorylates a number of nuclear proteins involved in DNA replication and transcription, and it is thought to play a pivotal role in nuclear events. We have found that DNA-PK forms a complex with autoantigen Ku (p70/p80) and ds DNA, and that autoantibodies to a 460 kDa catalytic polypeptide of DNA-PK (DNA-PKcs) are detected in patients with systemic rheumatic diseases. In this study, we have identified autoantibodies from various autoimmune diseases, developed in vitro kinase assay system to detect DNA-PK activity, and investigated the molecular mechanism of Ku protein and DNA-PKcs involved in Fas-mediated apoptosis using patient autoantibodies as probes.
In a survey of autoimmune sera, 11 sera were identified to contain autoantibodies to DNA-PK. Seven of 11 sera were accompanied with autoantibodies against to Ku protein, suggesting the antigen-driven mechanism might be involved in autoantibody production. Next, we utilized patient sera containing
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anti-DNA-PKcs antibodies to detect the DNA-PK activity. When the DNA-PKcs bound beads were incubated with the purified Ku protein, ds DNA and the synthetic peptide in the presence of [32P]ATP, a significant phosphorylation occured on the substrate. Apoptosis was induced by Jurkat cells in the presence of an agonistic anti-Fas antibody. The cleavage of DNA-PKcs to a 160 kDa fragment (p160) as well as 230 kDa fragment (p230) was observed. The cleavage was concomitant with an increase in CPP32. In immunoprecipitation assay, all autoimmune sera containing anti-DNA-PKcs antibodies immunoprecipitated p230 in addition to p160 after induction of apoptosis. To determine whether DNA-PKcs is targeted by apopain, we directly digested DNA-PKcs with recombinant apopain. When DNA-PKcs-Ku complex assembled on beads was treated with apopain, p230, p120, and p70/p80 were coprecipitated with anti-Ku antibodies and anti-DNA-PKcs antibodies even after complete digestion of DNA-PKcs, suggesting p230 and p120 were associated with Ku protein. Ku protein was not affected during Fas-mediated apoptosis.
We have clarified the clinical and immunological features of autoantibodies to DNA-PKcs Less
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