2001 Fiscal Year Final Research Report Summary
Study on the physiological function of synuclein family
| Project/Area Number |
11557196
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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 | 展開研究 |
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| Research Field |
医薬分子機能学
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| Research Institution | Showa University |
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Principal Investigator |
NAKAJO Shigeo Showa University, Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (50119236)
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| Co-Investigator(Kenkyū-buntansha) |
HIRABAYASHI Takahiro Showa University Center for Biotechnology, Instructor, 組換DNA実験室, 助手 (40297015)
SHIODA Seiji Showa University Medical School, Professor, 医学部, 教授 (80102375)
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| Project Period (FY) |
1999 – 2001
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| Keywords | synuclein / synuclein-binding protein / neuro-degeneration / transgenic mouse / 14-3-3 / metallothionein |
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| Research Abstract |
1) It was found that a-synudem is consiturtively phosphoiylated in both cells HEK 293 and FC12 which had been previously transfected with α-synudein gene. In both cell lines phosphorylalion was highly sensitive to phosphatases, since okedaic acid markedly stabilized phosphate incorporation. We have identified a major phosphorylation site at serine 129 which is phosphoiylated by casein kinases. It may be indicated that the function of α-synuciein is regulated by phosphorylation/dephosphorylation reaction.
2) β-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that β-synuclein protein was expressed within the cytoplasm and nudeus. Furthermore, β-synuclein immunoreadivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues.
3) We have prqared cDNA library fmm the cerebrum of reptiles (blue green snake) and amphibian (bullfrog) to clone cDNA of synudein family. Domain I of human β-synuclein
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was used as a prove. Ten positive clones have been obtained from the eDNA library of blue green snake. The sequence analysis is underadvance,
4) Using yeast two-hybrid system, we have identified 14-3-30 and metallothionein2A as β-synudein-binding protein. It has been shown that both proteins interact with β-synuclein in rat brain lysate, indicating that those interactions between β-synuclein and 14-3-30 and metallothionein2A occure even under the physiological conditions. Interestingly, it was also shown that β-synuclein bound to metallothionein3.
5) Transgenic mice using Cre-loxp system were produced to clarify the physiological function of α- and β-synucleins. In this experiment expression of synudeins is region specific in tissue since it is regulated by promotor used. We produced transgenic mice of two category, Cre-donor and Cre-acceptor mice. CaMkll promotor-Cre (hippocampus), GFAP promotor-Cre (astrocytes), Pgk2 promotor-Cre (testes), CAG promoter-Cre (whole body), Keratin14 piomotor-Cre (skin) transgenic mice were produced as Cre-dpnor, and loxP-neo-loxP (LNL)-cz-synudein and LNL-α-synuclein mice were as Cie-acceptor. Less
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