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The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death.
- Source :
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The Journal of biological chemistry [J Biol Chem] 2007 Sep 07; Vol. 282 (36), pp. 26562-74. Date of Electronic Publication: 2007 Jul 05. - Publication Year :
- 2007
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Abstract
- Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.
- Subjects :
- Amino Acid Substitution
Amyloid genetics
Animals
Apoptosis Regulatory Proteins genetics
Benzothiazoles
Binding Sites genetics
Cell Death drug effects
Cell Death genetics
Cysteine genetics
Glyceraldehyde-3-Phosphate Dehydrogenases genetics
HeLa Cells
Humans
Mutation, Missense
Neurons pathology
Oxidants pharmacology
Oxidation-Reduction drug effects
Protein Structure, Secondary genetics
Rabbits
Thiazoles metabolism
Amyloid metabolism
Apoptosis Regulatory Proteins metabolism
Cysteine metabolism
Glyceraldehyde-3-Phosphate Dehydrogenases metabolism
Neurons enzymology
Oxidative Stress genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 282
- Issue :
- 36
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17613523
- Full Text :
- https://doi.org/10.1074/jbc.M704199200