| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Heme is a prosthetic group of several enzymes, and essential for aerobic organisms. Since excess amount of heme in the cells increases reactive oxygen species, cellular heme level is strictly regulated under the balance of its biosynthesis and catabolism. To know how heme biosynthesis and catabolism is regulated by the mechanisms through protein-protein interaction, we have performed following experiments 1. To know what kind of proteins are involved in the regulation of heme biosynthesis and catabolism, we have screened human cDNA libraries using non-specific aminolevulinate synthase, erythroid-specific aminolevulinate synthase (ALAS2), heme oxygenase 1 or heme oxygenase 2 (HO-2) as a bait protein. As a result, we have found that one of the components of 20S proteasome could associate with HO-2 protein. Since over-expression of this protein reduced HO-2 protein expression level, this protein should be involved in the regulation of protein degradation of HO-2. 2. Using RNA interference
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(RNAi) technology, we have tried to establish the model cells for sideroblastic anemia. First, we have prepared effective small interfering RNA (siRNA) for suppression of ALAS2 expression, then, prepared expression vector, which express effective short-hairpin RNA (shRNA). This vector has been introduced into YN-1 cells, which is able to produce hemoglobin in the cells after the stimulation by transforming growth factor beta 1, and these cells were incubated with G418 to select the cells which constitutively express shRNA against ALAS2. YN-1 cells that express ALAS2 at low level were selected, and named as YN1-ALAS21ow cells. However, ringed sideroblast, which is the hallmark of the sideroblastic anemia, was not observed even after the erythroid differentiation by TGF-betal. We have confirmed that YN1-ALAS21ow cells expressed lower level of ALAS2 mRNA than control cells, YN1-ALAS21ow cells might expressed enough level of ALAS2 for protecting the cells from iron accumulation in mitochondria. Less
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