2005 Fiscal Year Final Research Report Summary
Molecular analysis of C.elegans AAA proteins related to genetic diseases and developmental disorders
| Project/Area Number |
16570147
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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 |
Molecular biology
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
YAMANAKA Kunitoshi KUMAMOTO UNIVERSITY, INSTITUE 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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| Co-Investigator(Kenkyū-buntansha) |
OGURA Teru KUMAMOTO UNIVERSITY, INSTITUE OF MOLECULAR EMBRYOLOGY AND GENETICS, PROFESSOR, 発生医学研究センター, 教授 (00158825)
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| Project Period (FY) |
2004 – 2005
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| Keywords | AAA protein / ATPase / C.elegans / hereditary spastic paraplegia / molecular chaperone / neurodegenerative disease / polyglutamine disease / p97 / VCP |
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| Research Abstract |
Caenorhabditis elegans possesses two homologues of p97/VCP/Cdc48p (CDC-48.1 and CDC-48.2), while humans and mice possess only one. To know their cellular functions, their mutant worms were analyzed. The cdc-48.1 deletion mutant showed slightly slow growth rate. Interestingly, we found that the cdc-48.1 deletion mutation decreased the brood size to half and concomitantly increased the number of non-fertilized eggs. Sperm in the cdc-48.1 mutant were exhausted at the stage earlier than that observed in the wild-type. These results together with the mating experiments suggest that C.elegans p97 functions in the switching processes from spermatogenesis to oogenesis. We also found that p97 plays a crucial role in unfolded protein response and ER network formation.
We found that spastin is involved in micritubule dynamics in vivo. We also found that purified spastin was able to directly bind tubulin in vitro and the ATPase activity of spastin was stimulated by tubiulin.
To elucidate the mechanism of ATP hydrolysis, we prepared several mutants of the C.elegans fidgetin homologue FIGL-1 carrying a mutation in each of three motifs, the Walker A and B motifs and the second region of homology. None of the constructed mutants showed ATPase activity. All the mutants except for K362A were able to bind ATP. Mixtures of E416A and R471A, or N461A and R471A led to the formation of hetero-hexamers with partially restored ATPase activities, providing direct, firm evidence for the intersubunit catalysis model. In addition, based on the results obtained with mixtures of K362A with wild-type or R471A subunits, we proposed that a conformational change upon ATP binding is required for proper orientation of the arginine fingers, which is essential for efficient hydrolysis of ATP bound to the neighboring subunit.
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