The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death.
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.
Nakajima
Hidemitsu
H
Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuen-cho, Naka-ku, Sakai City 599-8531, Japan. hnakajima@vet.osakafu-u.ac.jp
Amano
Wataru
W
Fujita
Akikazu
A
Fukuhara
Ayano
A
Azuma
Yasu-Taka
YT
Hata
Fumiaki
F
Inui
Takashi
T
Takeuchi
Tadayoshi
T
D019943:Amino Acid Substitution; D000682:Amyloid / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D052160:Benzothiazoles; D001665:Binding Sites / Q000235:genetics; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011817:Rabbits; D013844:Thiazoles / Q000378:metabolism
D000682:Amyloid; D051017:Apoptosis Regulatory Proteins; D052160:Benzothiazoles; D016877:Oxidants; D013844:Thiazoles; C009462:thioflavin T; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine