Molecular and cellular biology of C. elegans AAA proteins involved in human genetic diseases
| Project/Area Number |
14580684
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Molecular biology
|
|---|
| Research Institution | KUMAMOTO UNIVERSITY |
|---|
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)
|
|---|
| 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)
|
|---|
| Project Period (FY) |
2002 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Keywords | AAA protein / ATPase / C. elegans / hereditary spastic paraplegia / molecular chaperone / neurodegenerative disease / polyglutamine disease / p97 / VCP |
|---|
| Research Abstract |
RNAi assays revealed that simultaneous disruption of both p97/VCP homologues (C41C4.8 and C06A1.1) was embryonic lethal, although single disruption had no detectable defect, suggesting that their cellular functions are redundant. It is important to note that C. elegans possesses two highly similar homologues of p97/VCP, while human and mouse possess only one. The polyglutamine repeats were expressed as green fluorescent protein (GFP) -fusion proteins in the body wall muscle cells using the well-characterized unc-54 promoter. When the repeats were longer than 40, discrete cytoplasmic aggregates were formed and already appeared early in embryogenesis. The formation of aggregates did not affect on motility of young adult transgenic animals and on life span. Aggregate formation was partially but significantly suppressed by co-expression of C41C4.8, C06A1.1 or MAC-1. These suggest that p97/VCP homologues, AAA chaperones, play a crucial role in controlling polyglutamine aggregation.
The analysis of GFP fusion constructs of Y47G6A.10 and Y38F2AR.para that are paraplegin homologues showed that their expression pattern was not identical. In addition, effects of RNAi were also different ; mixed phenotype (embryonic lethal, larval lethal or slow growth) for Y47G6A.10, but no obvious effect for Y38F2AR.para. Progressively retarded motility was also observed for Y47G6A.10. Succinate dehydrogenase assay and electron microscopic observation of Y47G6A.10 (RNAi) indicated mitochondrial defects. These are in good agreement with the previous reports of the clinical characteristic of HSP patients and the analyses of muscle biopsy from patients, suggesting that Y47G6A.10 is a functional homologue of paraplegin.
Fidgetin protein was purified from recombinant baculovirus infected insect cells. Many mutant proteins were prepared and also purified. Detailed in vitro analysis of ATPase activity strongly supported the intersubunit catalytic mechanism for the ATP hydrolysis.
|
Report
(3 results)
Research Products
(12 results)
