title: Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan.
authors.forename: Takeya
authors.initials: T
authors.lastname: Kubo
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan. Electronic address: hnakajima@vet.osakafu-u.ac.jp.
authors.forename: Hidemitsu
authors.initials: H
authors.lastname: Nakajima
authors.affiliation: Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, Sakai, Osaka 5998531, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Nakatsuji
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Itakura
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kaneshige
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan.
authors.forename: Yasu-Taka
authors.initials: YT
authors.lastname: Azuma
authors.affiliation: Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, Sakai, Osaka 5998531, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Inui
authors.affiliation: Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuourai-kita, Izumisano, Osaka 5988531, Japan.
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Takeuchi
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000494:pharmacology*; D066329:Protein Aggregates / Q000187:drug effects; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D066329:Protein Aggregates; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine
keywords: Dominant-negative; GAPDH; Nitric oxide; Protein aggregation; Therapeutics