Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions

Abstract

Knowledge of the cellular targets of ROS (reactive oxygen species) and their regulation is an essential prerequisite for understanding ROS-mediated signalling. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is known as a major target protein in oxidative stresses and becomes thiolated in its active site. However, the molecular and functional changes of oxidized GAPDH, the inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by two-dimensional gel electrophoresis and identified them using nanoLC-ESI-q-TOF MS/MS (nano column liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem MS). Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on two-dimensional gel electrophoresis and were identified as cysteine disulfide and cysteic acids on Cys152 in the active site. We identified the interacting proteins of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNA-binding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor), both of which are known to exist as heterodimers and bind to RNA and DNA. Interaction between oxidized GAPDH and p54nrb was abolished upon expression of the GAPDH active site mutant C152S. The C-terminal of p54nrb binds to GAPDH in the cytosol in a manner dependent on the dose of hydrogen peroxide. The GAPDH-p54nrb complex enhances the intrinsic topoisomerase I activation by p54nrb-PSF binding. These results suggest that GAPDH exerts other functions beyond glycolysis, and that oxidatively modified GAPDH regulates its cellular functions by changing its interacting proteins, i.e. the RNA splicing by interacting with the p54nrb-PSF complex.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

“…GAPDH was also reported to be modified under oxidative stress conditions. Specifically, a cysteine residue in the active site of GAPDH was oxidized to cysteic acid, which makes this enzyme inactive (47,48). Although the precise mechanism by which GAPDH is translocated to mitochondria has not yet been determined, oxidative stress in I/R potentially regulates the onset of GAPDH and PKC␦-mediated mitophagy.…”

Integration and Weighing of Omics Data for Obesity

“…Hwang et al [79] study acknowledges that an important oxidative target of ROS is GAPDH. One of the consequences of oxidative stress is a decrease in cellular ATP levels and blocked glycolysis [80,81], due to the inactivation of the glycolytic enzyme GAPDH.…”

An improved analysis methodology for translational profiling by microarray

“…Among the established concepts, the category "Phosphorus metabolic process," cross-referenced to the highest number of publications, was identified specifically from the INCATome methodology. PSF has been shown to regulate gene expression of mitochondrial phosphate carrier (PiC) as well as interact with oxidatively modified GAPDH (Iacobazzi et al 2005;Hwang et al 2009). Furthermore, two out of three emerging concepts were revealed by INCATome and all hinted toward a role in adhesion.…”