2003 Fiscal Year Final Research Report Summary
Structural and functional studies on AAA proteins in E. coli and C. elegans.
| Project/Area Number |
13480232
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Kumamoto University |
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Principal Investigator |
OGURA Teru KUMAMOTO UNIVERSITY, INSTITUTE OF MOLECULAR EMBRYOLOGY AND GENETICS, PROFESSOR, 発生医学研究センター, 教授 (00158825)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANAKA Kunitoshi KUMAMOTO UNIVERSITY, INSTITUTE OF MOLECULAR EMBRYOLOGY AND GENETICS, ASSISTANT PROFESSOR, 発生医学研究センター, 助教授 (90212290)
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| Project Period (FY) |
2001 – 2003
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| Keywords | E. coli / C. elegans / chaperone / ATPase / AAA protein / mitochondria / hereditary spastic paraplegia / polyglutamine disease |
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| Research Abstract |
We have studied on the AAA protease, FtsH, in E. coil and several AAA proteins in C. elagans, and have obtained the following results.
FtsH protease
1.The crystal structure of the ATPase domain of FtsH was determined. A hexameric model of the ATPase domain supports an inter-subunit catalysis model for ATP hydrolysis.
2.FtsH contains highly conserved aromatic and glycine residues in the central pore region of the hexamer. We have shown that these residues have important roles in proteolysis and its coupling to ATP hydrolysis.
3.Mutations in acidic residues located in the central channel affected proteolysis and ATPase activity.
4.We have established a fluorescence polarization assay system to monitor substrate degradation spectrometrically. Using the system, we have determined the direction of substrate degradation and the energy cost of proteolysis.
AAA proteins in C. elegans
1.RNAi assays for paraplegin homologs revealed mixed phenotype (embryonic lethal, larval lethal and slow growth) for Y47G6A.10, but no obvious effect for Y38F2AR.para. Progressively retarded motility was also observed for Y47G6A.10. Histochemical and EM analyses indicated mitochondrial defects.
2.C. elegans has two p97/VCP homologs, We have shown that these homologs have essential but redundant functions.
3.We have expressed polyQ expansions fused to GFP in the body wall muscle cells. When the repeats were longer than 40, discrete cytoplasmic aggregates were formed. The formation of aggregates was partially suppressed by co-expression of either p97 homolog.
4.A homolog of fidgetin is essential for gonadogenesis in C. elegans. Recombinant fidgetin proteins were purified and assayed for ATPase activity. In vitro analysis of mutant proteins further supported the inter-subunit catalysis mechanism for ATP hydrolysis by AAA proteins.
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[Publications] Krzywda, S., Brzozowski, A.M., Verma, C., Karata, K., Ogura, T., Wilkinson, A.J.: "The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coil at 1.5 Å resolution."Structure. 10. 1073-1083 (2002)
Description
「研究成果報告書概要(欧文)」より
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