TY - JOUR
T1 - Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions
AU - Glover, Mia R.
AU - Davies, Michael J.
AU - Fuentes-Lemus, Eduardo
N1 - Publisher Copyright:
© 2023 The Authors
PY - 2024
Y1 - 2024
N2 - Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and ‘moonlighting’ activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.
AB - Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and ‘moonlighting’ activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.
KW - Glyceraldehyde-3-phosphate dehydrogenase
KW - Hydrogen peroxide
KW - Macromolecular crowding
KW - Peroxynitrite
KW - Redox signaling
KW - Thiol oxidation
UR - http://www.scopus.com/inward/record.url?scp=85180344665&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2023.12.015
DO - 10.1016/j.freeradbiomed.2023.12.015
M3 - Journal article
C2 - 38122871
AN - SCOPUS:85180344665
VL - 212
SP - 1
EP - 9
JO - Free Radical Biology & Medicine
JF - Free Radical Biology & Medicine
SN - 0891-5849
ER -