Elsevier

Nitric Oxide

Volume 53, 29 February 2016, Pages 13-21
Nitric Oxide

Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death

https://doi.org/10.1016/j.niox.2015.12.005Get rights and content
Under a Creative Commons license
open access

Highlights

  • The active site cysteine-152 of GAPDH is critical for its aggregation.

  • C152A-GAPDH interferes with nitric oxide-induced GAPDH aggregation.

  • C152A-GAPDH restores nitric oxide-induced cell death.

  • A heterotetramer of wild-type- and C152A-GAPDH provides key interference.

  • We outline a therapeutic strategy for GAPDH aggregation-mediated 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.

Keywords

GAPDH
Nitric oxide
Protein aggregation
Dominant-negative
Therapeutics

Abbreviations

ADH
alcohol dehydrogenase
AFM
atomic force microscopy
ALD
aldolase
DOX
doxycycline
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
GSH
glutathione
MDH
malate dehydrogenase
Mw
molecular weight
NO
nitric oxide
NOC18
1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene
NOR3
(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide
PBS
phosphate-buffered saline
PMSF
phenylmethylsulfonyl fluoride
WT
wild type

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