Analysis of protective roles for renal injury by heat shock proteins treansfected with HSP genes
| Project/Area Number |
14571011
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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 |
Kidney internal medicine
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| Research Institution | Akita University |
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Principal Investigator |
KOMATSUDA Atsushi Akita University, School of Medicine, Assistant Professor, 医学部, 講師 (70272044)
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| Co-Investigator(Kenkyū-buntansha) |
WAKUI Hideki Akita University, School of Medicine, Assistant Professor, 医学部, 講師 (70240463)
ITOH Hideaki Akita University, Faculty of Engineering and Resource Science, Professor, 工学資源学部, 教授 (80168369)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | heat shock proteins / HSP73 / gentamicin / chaperon / renal toxicity / HSP60 / HSP105 / dehydration / HSP72 / HSP90 / シャペロン / 遺伝子導入 / 耐性 / 浸透圧 |
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| Research Abstract |
We investigated HSP60 import system. From immunoblotting and immunohistochemistry, HSP60 was detected in both cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial assay. HSP60 mRNA was increased in the renal papilla of rats that had been restricted water for three and five days, but no changes in HSP60 mRNA in the renal cortex or medulla. On immunoblotting, HSP60 was detected in both cytoplasm and mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the renal papilla of rats with water restriction, but the protein was increased in the mitochondria of the rat renal papilla. We also analyzed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from th
… More
e affinity column by an excess of signal peptide or by 5 mM ATP. On immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytosolic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP60.
We have characterized the biochemical properties of the testis and brain specific 105-kDa protein, which is cross-reacted with an anti-bovine HSP90 antibody. The 105-kDa protein inhibited the aggregation of citrate synthase as a molecular chaperone in vitro. ATP/MgCl2 has slightly influenced the suppression of the citrate synthase aggregation by 105-kDa protein. The protein was able to bind to ATP-Sepharose like the other molecular chaperone HSP70. A partial amino-acid sequence (24 amino-acid residues) of the protein was determined and coincided with those of the mouse testis-and brain-specific APG-1 and osmotic stress protein 94 (OSP94). The 105-kDa protein was detected only in the renal medulla similar to OSP94 upon immunoblotting. The purified 105-kDa protein was cross-reacted with an antibody against APG-1. These results suggested that APG-1 and OPS94 are both identical to the 105-kDa protein.
Gentamicin (GM) has been used widely as an antibiotic. However, the specific binding protein of the drug has not yet been understood sufficiently. We investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the elutant. By immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known as a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury. Less
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Report
(4 results)
Research Products
(10 results)
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[Journal Article] 73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin.2004
Author(s)
Miyazaki T, Sagawa R, Honma T, Noguchi S, Harada T, Komatsuda A, Ohtani H, Wakui H, Sawada KI, Otaka M, Watanabe S, Jikei M, Ogawa N, Hamada F, Itoh H.
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Journal Title
J Biol Chem 279(17)
Pages: 17295-17300
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Characterization of the 105-kDa molecular chaperone. Identification, biochemical properties, and localization.2002
Author(s)
Matsumori M, Itoh H, Toyoshima I, Komatsuda A, Sawada K, Fukuda J, Tanaka T, Okubo A, Kinouchi H, Mizoi K, Hama T, Suzuki A, Hamada F, Otaka M, Shoji Y, Takada G.
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Journal Title
Eur J Biochem 269 (22)
Pages: 5632-5641
Description
「研究成果報告書概要(欧文)」より
Related Report
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