2004 Fiscal Year Final Research Report Summary
Isolation of the interacting proteins with aprataxin and its subcellular localization.
| Project/Area Number |
15590905
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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 |
Neurology
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| Research Institution | JICHI MEDICAL SCHOOL |
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Principal Investigator |
SHIMAZAKI Haruo Jichi Medical School, Faculty of medicine, assistant professor, 医学部, 助手 (30316517)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIYAMA Yoshihisa Jichi Medical School, Faculty of medicine, lecturer, 医学部, 講師 (00245052)
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| Project Period (FY) |
2003 – 2004
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| Keywords | EAOH / aprataxin / subcellular localization / interacting proteins |
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| Research Abstract |
Aprataxin is the causative protein for early-onset ataxia with ocular motor apraxia and hypoalbuminemia(EAOH). To elucidate the onset mechanism of EAOH, we studied the subcellular localization of aprataxin in cultured cells, and isolated interacting proteins with it.
We transfected expression vectors of EGFP or myc-tagged aprataxin into COS7 cells. After 48 hours incubation, we observed the cells with confocal laser microscopy. The wild type short-form aprataxin was mainly distributed to cytoplasm, whereas mutant short-form aprataxins (T insertion at nt 167, A to G transition at nt 80 and C to T transition at nt 95) were localized to nucleus. Both wild type and mutant (A to G transition at nt 80) long-form aprataxins were observed in the nucleus. Mutant short-form aprataxin would change the cellular localization because the insertion mutation induced trancation of aprataxin, and the misssence mutation on the histidine trial domain altered the function of aprataxin. These results suggested that short-form aprataxin was mainly concerned about the onset mechanism of EAOH. We will observe the cells expressed other mutation-carried long-form aprataxin.
We tried to identify the interacting proteins with short-form aprataxin using bacterial two-hybrid method. We use short-form aprataxin as bait, and screened against fetal brain cDNA library. Two proteins were isolated. One protein was collapsin response mediated protein-2(CRMP-2). CRMP-2 contributes the axonal guidance and polarity of neurons. Thus CRMP-2 dysfunction might result in the peripheral neuropathy of EAOH. The other protein was the product of FLJ31106 (cDNA). This protein was not characterized, but had homology to HSPC166, expressed in CD34-positive hematopoetic stem cells. We will confirm these interactions with immunoprecipitation method.
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