2006 Fiscal Year Final Research Report Summary
A novel mechanism for the regulation of cellular apoptosis mediated by posttranslational N-myristoylation of cytoskeletal proteins.
| Project/Area Number |
17580080
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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 |
Applied biochemistry
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| Research Institution | Yamaguchi University |
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Principal Investigator |
UTSUMI Toshihiko Yamaguchi University, Graduate School of Medicine, Professor, 大学院医学系研究科, 教授 (20168727)
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| Project Period (FY) |
2005 – 2006
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| Keywords | posttranslational modification / protein N-myristoylation / gelsolin / Cytoskeletal protein / apoptosis posttranslational / N-myristoylation |
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| Research Abstract |
Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could also occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal 9 residues of the C-terminal caspase-cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation.
To analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase-cleavage products expressed in COS-1 cells with [^3H]myristic acid was performed. It was found that the C-terminal caspase-cleavage product of human gelsolin (tGelsolin) expressed in COS-1 cells was efficiently N-myristoylated. When COS-1 cells transiently transfected with cDNA coding for full-length gelsolin were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was gener
… More
ated, as demonstrated by in vivo metabolic labeling. The caspase-mediated generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressd in Hela cells. Immunofluorescence staining (coupled with MitoTracker staining) and subcellular fractionation revealed that exogenously expressed tGelsolin did not localize to mitochondria, but rather was diffusely distributed in the cytoplasm.
To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using the plasmid tGelsolin-IRES-EGFP significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis.
These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin. Less
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[Journal Article] Preparation of N-acylated proteins modified with fatty acids having a specific chain length using an insect cell-free protein synthesis system.2007
Author(s)
Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., Tsunasawa, S., Nishimura, 0.
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Journal Title
Biosci. Biotech. Biochem. 71
Pages: 261-264
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Detection of co-and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system.2007
Author(s)
Sakurai, N., Moriya, K., Suzuki, T., Sofuku, K., Mochiki, H., Nishimura, 0., Utsumi, T.
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Journal Title
Anal. Biochem. 362
Pages: 236-244
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] N-Terminal protein modifications in an insect cell-free protein synthesis system and their identifi-cation by mass spectrometry.2006
Author(s)
Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., Tsunasawa, S., Nishimura, 0.
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Journal Title
Proteomics 6
Pages: 4486-4495
Description
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[Journal Article] 4-Hydroxy-3,5,3',4'-Tetrachlorobiphenyl induced membrane permeability transition in isolated rat liver mitochondria.2006
Author(s)
Fujita, H., Okimura, Y., Utsumi, T., Kitamura, S., Kuroki, H., Otsuki, T., Sasaki, J., Kashiwagi, A., Utsumi, K.
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Journal Title
J. Clin. Biochem. Nutr. 38
Pages: 167-175
Description
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[Journal Article] Involvement of Ras/ extra-cellular signal-regulated kinase, but not Akt pathway in risedronate-induced apoptosis of U937 cells and its suppression by cytochalasin B.2005
Author(s)
Fujita, H., Utsumi, T., Muranaka, S., Ogino, T., Yano, H., Akiyama, J., Yasuda, T., Utsumi, K.
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Journal Title
Biochem Pharmacol. 69
Pages: 1773-1784
Description
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[Journal Article] Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry.
Author(s)
Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., Tsunasawa, S., Nishimura, 0.
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Journal Title
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