Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions.
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.
Hwang
Na Rae
NR
Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Department of Bioinspired Science, Ewha Womans University, Seoul, South Korea.
Yim
Seung-Hee
SH
Kim
Young Mee
YM
Jeong
Jaeho
J
Song
Eun Joo
EJ
Lee
Yoonji
Y
Lee
Jin Hee
JH
Choi
Sun
S
Lee
Kong-Joo
KJ
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008958:Models, Molecular; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D050810:Octamer Transcription Factors / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016601:RNA-Binding Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D016601:RNA-Binding Proteins; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine