Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin-Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

The amyloid precursor protein affects glyceraldehyde 3-phosphate dehydrogenase levels, organelle localisation and thermal stability.

Glyceraldehyde 3-phosphate dehydrogenase's (GAPDH) proapoptotic response to cellular oxidative stress has suspected implication for Alzheimer's disease (AD). Interestingly, the overexpression of the amyloid precursor protein (APP) can initiate oxidative stress responses within mammalian cell lines. Here, APP695 and APP770 overexpression significantly increased the level of GAPDH, while no effect was observed when the APP homologues APLP1 or APLP2 were used. Heterologous expression of APP695 was shown to increase the level of GAPDH within the cytoplasm by over 100% and within the mitochondria by approximately 50%. Moreover, a shift in organelle distribution from cytoplasm > nucleus > mitochondria in control cell lines to cytoplasm > mitochondria > nucleus in the APP695 overexpressing cell line was also observed. Further, the overexpression of APP695 increased GAPDH aggregation temperature by 3.09 ± 0.46 °C, indicative of greater thermal stability. These results demonstrate a clear correlation between APP overexpression and GAPDH levels, organelle distribution and thermal stability.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight.

KEY MESSAGE: MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Sperm-Specific Glyceraldehyde-3-Phosphate Dehydrogenase - An Evolutionary Acquisition of Mammals.

This review is focused on the mammalian sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). GAPDS plays the major role in the production of energy required for sperm cell movement and does not perform non-glycolytic functions that are characteristic of the somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. The GAPDS sequence is composed of 408 amino acid residues and includes an additional N-terminal region of 72 a.a. that binds the protein to the sperm tail cytoskeleton. GAPDS is present only in the sperm cells of mammals and lizards, possibly providing them with certain evolutionary advantages in reproduction. In this review, studies concerning the problems of GAPDS isolation, its catalytic properties, and its structural features are described in detail. GAPDS is much more stable compared to the somatic isoenzyme, perhaps due to the necessity of maintaining the enzyme function in the absence of protein expression. The site-directed mutagenesis approach revealed the two GAPDS-specific proline residues, as well as three salt bridges, which seem to be the basis of the increased stability of this protein. As distinct from the somatic isoenzyme, GAPDS exhibits positive cooperativity in binding of the coenzyme NAD+. The key role in transduction of structural changes induced by NAD+ is played by the salt bridge D311-H124. Disruption of this salt bridge cancels GAPDS cooperativity and twofold increases its enzymatic activity instead. The expression of GAPDS was detected in some melanoma cells as well. Its role in the development of certain pathologies, such as cancer and neurodegenerative diseases, is discussed.

BDNF modulates contextual fear learning during adolescence.

Brain-derived neurotrophic factor (BDNF) is a growth factor that plays key roles in regulating higher-order emotional and cognitive processes including fear learning and memory. A common single-nucleotide polymorphism (SNP) has been identified in the human BDNF gene (BDNF Val66Met) that leads to decreased BDNF secretion and impairments in specific forms of fear learning in adult humans and genetically modified mice containing this SNP. As the emergence of anxiety and other fear-related disorders peaks during adolescence, we sought to better understand the impact of this BDNF SNP on fear learning during the transition through adolescence in BDNF Val66Met knock-in mice. Previously, we have shown that contextual fear expression is temporarily suppressed in wild-type mice during a distinct period in adolescence, but re-emerges at later, postadolescent ages. Until recently, it was unclear whether BDNF-TrkB signaling is involved in the modulation of hippocampal-dependent contextual fear learning and memory during this adolescent period. Here we show that in BDNF Val66Met mice, the presence of the Met allele does not alter contextual fear expression during adolescence, but when previously conditioned BDNF(Met/Met) mice are tested in adulthood, they fail to display the delayed expression of contextual fear compared to wild-type BDNF(Val/Val) controls, indicating that the Met allele may permanently alter hippocampal function, leading to persistent functioning that is indistinguishable from the adolescent state. Conversely, truncated TrkB receptor (TrkB.T1)-deficient (TrkB.T1(-/-)) mice, a genetic mouse model with increased BDNF-TrkB signaling through full-length TrkB receptors, exhibit an accelerated expression of contextual fear during adolescence compared to wild-type controls. Our results point to a critical function for BDNF-TrkB signaling in fear regulation in vivo, particularly during a potentially sensitive period in adolescence.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer's disease.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme that catalyzes the sixth step of glycolysis and thus, serves to break down glucose for energy production. Beyond the traditional aerobic metabolism of glucose, recent studies have highlighted additional roles played by GAPDH in non-metabolic processes, such as control of gene expression and redox post-translational modifications. Neuroproteomics have revealed high affinity interactions between GAPDH and Alzheimer's disease-associated proteins, including the ß-amyloid, ß-amyloid precursor protein and tau. This neuronal protein interaction may lead to impairment of the GAPDH glycolytic function in Alzheimer's disease and may be a forerunner of its participation in apoptosis. The present review examines the crucial implication of GAPDH in neurodegenerative processes and clarifies its role in apoptotic cell death.

Functional diversity.

There is increasing evidence to support a gene economy model that is fully based on the principles of evolution in which a limited number of proteins does not necessarily reflect a finite number of biochemical processes. The concept of 'gene sharing' proposes that a single protein can have alternate functions that are typically attributed to other proteins. GAPDH appears to play this role quite well in that it exhibits more than one function. GAPDH represents the prototype for this new paradigm of protein multi-functionality. The chapter discusses the diverse functions of GAPDH among three broad categories: cell structure, gene expression and signal transduction. Protein function is curiously re-specified given the cell's unique needs. GAPDH provides the cell with the means of linking metabolic activity to various cellular processes. While interpretations may often lead to GAPDH's role in meeting focal energy demands, this chapter discusses several other very distinct GAPDH functions (i.e. membrane fusogenic properties) that are quite different from its ability to catalyze oxidative phosphorylation of the triose, glyceraldehyde 3-phosphate. It is suggested that a single protein participates in multiple processes in the structural organization of the cell, controls the transmission of genetic information (i.e. GAPDH's involvement may not be finite) and mediates intracellular signaling.

[Regulation of intracellular signal pathways via sensor proteins by oxidative stress].

It has been known that reactive oxygen species (ROS) control the enzymatic and transcriptional activity of proteins via direct modification of cysteine residues. Hence, oxidation of cysteine thiol could be a vital modulator of signal transduction pathways. These findings indicate that some proteins serve as the sensor proteins highly sensitive to ROS. In this review, I show the relationship between intracellular ROS sensor and the regulation of protein function via oxidation.

GAPDH: a common enzyme with uncommon functions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as an important enzyme for energy metabolism and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm. Recent studies have shown that GAPDH has multiple functions independent of its role in energy metabolism. Although increased GAPDH gene expression and enzymatic function is associated with cell proliferation and tumourigenesis, conditions such as oxidative stress impair GAPDH catalytic activity and lead to cellular aging and apoptosis. The mechanism(s) underlying the effects of GAPDH on cellular proliferation remains unclear, yet much evidence has been accrued that demonstrates a variety of interacting partners for GAPDH, including proteins, various RNA species and telomeric DNA. The present mini review summarizes recent findings relating to the extraglycolytic functions of GAPDH and highlights the significant role this enzyme plays in regulating both cell survival and apoptotic death.

The glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase and enolase interact with the renal epithelial K+ channel ROMK2 and regulate its function.

BACKGROUND/AIMS: ROMK channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. The aim was to study the functional implications of the interaction between ROMK2 (Kir1.1b) and two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase-α, which were identified as potential regulatory subunits of the channel complex. METHODS: We performed a membrane yeast-two-hybrid screen of a human kidney cDNA library with ROMK2 as a bait. Interaction of ROMK2 with GAPDH and enolase was verified using GST pull-down, co-immunoprecipitation, immunohistochemistry and co-expression in Xenopus oocytes. RESULTS: Confocal imaging showed co-localisation of enolase and GAPDH with ROMK2 in the apical membrane of the renal epithelial cells of the thick ascending limb. Over-expression of GAPDH or enolase-α in Xenopus oocytes markedly reduced the amplitude of ROMK2 currents but did not affect the surface expression of the channels. Co-expression of the glycolytically inactive GAPDH mutant C149G did not have any effect on ROMK2 current amplitude. CONCLUSION: Our results suggest that the glycolytic enzymes GAPDH and enolase are part of the ROMK2 channel supramolecular complex and may serve to couple salt reabsorption in the thick ascending limb of the loop of Henle to the metabolic status of the renal epithelial cells.

Internal initiation of translation.

Although the 5' cap-dependent scanning mechanism can account for the translational initiation of most mRNAs in eukaryotic cells, several viral and cellular mRNAs contain nucleotide sequences in their 5' non-coding regions that can mediate binding of ribosomes to the mRNA, regardless of the modification state of the 5' ends. During the past year, some nuclear proteins normally involved in RNA processing have been shown also to facilitate 'internal' ribosome binding. Unexpected dual functions have, therefore, been suggested for these RNA-binding proteins, in both RNA biogenesis in the nucleus and RNA translation in the cytoplasm.

Oxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility.

The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5-3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.

Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity.

Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.

[Calcium pros and cons significance and risk of phosphorus supplementation. The necessity of phosphorus supplementation for hypophosphatemia].

Phosphate plays a vital role forming the high-energy band within ATP. The pathophysiological results of phosphate deficiency are inadequate supplies of energy-rich phosphates and, in particular, inhibition of glyceraldehyde-3- phosphate dehydrogenase, which occupies a key position in glycolysis. The effect of this on the central nervous system, muscle and erythrocyte energy metabolism is to reduce ATP and 2,3-diphosphoglycerate levels, leading to left-hand displacement of the oxygen-hemoglobin dissociation curve with decreased peripheral oxygen uptake and transport. Therefore, detection and treatment of acute hypophosphatemia is important in many hospitalized patients particularly in ICU patients. Severe hypophosphatemia is also associated with a number of neuromuscular and cardiovascular sequelae, in which phosphate supplementation leads to improved symptoms and clinical parameters. In clinical practice it is common on administering 0.4 mmol (12 mg) phosphate/kg per day, and to adjust this on the basis of the serum phosphate analysis.

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates.

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.

The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

Interstitial flow induces MMP-1 expression and vascular SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism.

The migration of vascular smooth muscle cells (SMCs) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have previously shown that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression, which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor PD-98059 completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation, whereas PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of activator protein-1 transcription factors (c-Jun and c-Fos), whereas PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, whereas silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggest that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.

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.