| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Keiko Kagoshima University, Graduate School of Medical and Dental Sciences, Associate Professor, 大学院・医歯学総合研究科, 助教授 (70108869)
SAHEKI Takeyori Kagoshima University, Graduate School of Medical and Dental Sciences, Professor, 大学院・医歯学総合研究科, 教授 (10056070)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Adult-onset type II citrullinemia(CTLN2)is an autosomal recessive disease caused by mutations in SLC25A13 located on chromosome 7q21.3.The gene encodes a polypeptide of 675 amino acids, designated citrin, which is a liver-type of calcium-binding mitochondrial aspartate glutamate carrier(AGC).Previously, we have found fragmentation of mitochondria in hepatoma cells overexpressed fusion protein with the mutated citrin and green fluorescence protein(GFP).
1.The subcellular localization analysis using green fluorescence revealed that wild type citrin-GFP was localized in the mitochondria, but the mutated citrins([VI], [VII], [VIII]and[IX]), which have some aberrations in the C-terminal end located outside the last transmembrane spanner, were localized in both mitochondria and cytoplasm.Proteoliposome assay showed that the mutants[VIII]and[IX]had no AGC activity, but AGC activity in mutant[VII]was 60% of wild-type citrin.These results suggest that the length(4 amino acids)of the C-end is req
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uired for AGC activity and glutamate at position 601 plays an important role in the AGC activity of citrin.In the liver of CTLN2 patient with mutation[VII], no cross-reactive immune material was detected by Western blot analysis with anti-human citrin antibody, suggesting that the mutant[VII] citrin may be less stable in the liver of CTLN2 patients.
2.To investigate the possibility of cell death initiated by the mutated citrin which induced swelling and fragmentation of mitochondria, we performed immunohistochemical analysis of cytochrome c release in the mutated citrin transfected cells.However, we have not had any positive results on cytochrome c release from mitochondria to cytoplasm.On the other hand, we found that levels of plasma cytochrome c in the patients with citrin eficiency were high in neonate but low in other period, although more detailed experiments are required.
3.We generated citrin-KO mice which defected slc25a13 mRNA, citrin protein, and activities in aspartate glutamate shuttle and in ureogenesis from ammonia.However, up to 1 year of age, citrin-KO mice failed to show CTLN2-like symptoms.The rate of protein synthesis in primary hepatocytes of citrin-KO mice was the same as control.These results suggest that mice have some compensation system(s). Less
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