1999 Fiscal Year Final Research Report Summary
Molecular accumulation mechanisms of small heat shock proteins in neural tissues.
| Project/Area Number |
09680785
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Aichi Human Service Center, Institute for Developmental Research |
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Principal Investigator |
INAGUMA Yutaka Aichi Human Service Center, Institute for Developmental Research, Department of Biochemistry, Researcher, 生化学部, 主任研究員 (10250250)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Kanefusa Aichi Human Service Center, Institute for Developmental Research, Department of Biochemistry, Vice-president and Department head, 生化学部, 副所長兼部長 (50022801)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Alexander's disease / hsp / phosphorylation / two-hybrid / Ubc9 / sentrin / Drosopohila / mitochondria |
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| Research Abstract |
In order to elucidate the mechanisms of the accumulation of the small heat shock proteins in several neural diseases, we studied about (1) Phosphorylation of alpha B-crystallin, (2) Interactive partners of small heat shock proteins, (3) Mitochondrial distribution of Drosophila Hsp22. In addition, we analyzed a juvenile type of Alexander's disease using specific antibodies against phosphorylated alpha B-crystallin.
Three serine residues in alpha B-crystallin were phosphorylated under the various stress conditions. We generated affinity purified antibodies specifically recognized the each phosphorylation sites (p19S, p45S, and p59S). The anti-p45S antibodies recognized mitotic cells in immunocytochemistry and immunohistochemistry. We newly found the anti-p59S antibodies recognized the centrosome and midbody of dividing cells.
When the centrosomes were preincubated with antibodies against C-terminal peptide, andi-p45S antibodies, or anti-p59S antibodies, polymerization of microtubules were
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inhibited. Alpha B-crystallin might be involved in the mechanisms of chromsomal segregation.
Recent works have shown that the small heat shock proteins (sHsp) have chaperone-like activity. However, the nature of the target molecule has not been elucidated. We recently isolated Ubc9, a member of ubiquitin conjugating enzymes, from a Drosophila cDNA library by using yeast two-hybrid system, with sHsp as a bait. Ubc9 conjugates ubiquitin-like protein, sentrin-1 to certain proteins for intracellular compartmentalization. In order to understand which proteins are involved in this system we isolated human sentrin-1 cDNA by RT-PCR and generated an affinity-purified anti-sentrin antibody. We carried out immunohistochemistry and immunocytochemistry with this antibody in addition to biochemical analysis.
Immunoprecipitation showed that DmHsp22 did not interact with the other sHsps. In fact this sHsp was localized in mitochondria where it was found as an oligomeric structure in the matrix fraction. This localization was further confirmed using a DmHsp22-GFP construct. Less
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