Glyceraldehyde-3-phosphate Dehydrogenase Proteins Research Articles

Genome-Wide Characterization of Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Their Expression Profile under Drought Stress in Quercus rubra.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is crucial in plant metabolism and responses to various abiotic stresses. In the glycolysis pathway, glyceraldehyde-3-phosphate (G3P) is oxidized to 1,3-bisphosphate glycerate (1,3-BPG) through the catalytic action of GAPDH. However, the GAPDH gene family in Quercus rubra has been minimally researched. In this study, we identified 13 GAPDH-encoding genes in Q. rubra through a bioinformatics analysis of genomic data. Evolutionary studies suggest that these QrGAPDH genes are closely related to those in Glycine max and Triticum aestivum. We conducted a comprehensive whole-genome study, which included predictions of subcellular localization, gene structure analysis, protein motif identification, chromosomal placement, and analysis of cis-acting regions. We also examined the expression of GAPDH proteins and genes in various tissues of Q. rubra and under drought stress. The results indicated diverse expression patterns across different tissues and differential expression under drought conditions. Notably, the expression of Qurub.02G290300.1, Qurub.10G209800.1, and Qrub.M241600.1 significantly increased in the leaf, stem, and root tissues under drought stress. This study provides a systematic analysis of QrGAPDH genes, suggesting their pivotal roles in the drought stress response of trees.

Identification and expression profiling of GAPDH family genes involved in response to Sclerotinia sclerotiorum infection and phytohormones in Brassica napus.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a crucial enzyme in glycolysis, an essential metabolic pathway for carbohydrate metabolism across all living organisms. Recent research indicates that phosphorylating GAPDH exhibits various moonlighting functions, contributing to plant growth and development, autophagy, drought tolerance, salt tolerance, and bacterial/viral diseases resistance. However, in rapeseed (Brassica napus), the role of GAPDHs in plant immune responses to fungal pathogens remains unexplored. In this study, 28 genes encoding GAPDH proteins were revealed in B. napus and classified into three distinct subclasses based on their protein structural and phylogenetic relationships. Whole-genome duplication plays a major role in the evolution of BnaGAPDHs. Synteny analyses revealed orthologous relationships, identifying 23, 26, and 26 BnaGAPDH genes with counterparts in Arabidopsis, Brassica rapa, and Brassica oleracea, respectively. The promoter regions of 12 BnaGAPDHs uncovered a spectrum of responsive elements to biotic and abiotic stresses, indicating their crucial role in plant stress resistance. Transcriptome analysis characterized the expression profiles of different BnaGAPDH genes during Sclerotinia sclerotiorum infection and hormonal treatment. Notably, BnaGAPDH17, BnaGAPDH20, BnaGAPDH21, and BnaGAPDH22 exhibited sensitivity to S. sclerotiorum infection, oxalic acid, hormone signals. Intriguingly, under standard physiological conditions, BnaGAPDH17, BnaGAPDH20, and BnaGAPDH22 are primarily localized in the cytoplasm and plasma membrane, with BnaGAPDH21 also detectable in the nucleus. Furthermore, the nuclear translocation of BnaGAPDH20 was observed under H2O2 treatment and S. sclerotiorum infection. These findings might provide a theoretical foundation for elucidating the functions of phosphorylating GAPDH.

The variations in gene expression of GAPDH in Ocimum basilicum cultivars under drought-induced stress conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) holds a pivotal role within the glycolytic pathway of higher plants. It has garnered attention as a significant target protein in instances of oxidative stress, where it can engage in thiolation reactions within its active site. Numerous genes encoding cytosolic iterations of GAPDH have been identified and analyzed in specific plant species. This investigation was conducted to gain insights into GAPDH's function amidst drought-induced stress. Within this framework, the basil plant (Ocimum basilicum) was chosen for focused exploration, encompassing the cloning of the comprehensive cDNA of basil GAPDH (ObGAPDH) and scrutinizing its patterns of expression. The complete sequence of Ob-GAPDH spanned 1315 base pairs. The resultant protein derived from this sequence comprised 399 amino acids, projecting a molecular weight of approximately 42.54kDa and an isoelectric point (pI) of 6.01. An examination of the evolutionary connections among various GAPDH proteins unveiled ObGAPDH's shared lineage with GAPDH proteins sourced from other plants, such as Salvia splendens and Sesamum indicum. Furthermore, computational methodologies were harnessed to predict the potential oxidative role of ObGAPDH in response to external signals. Molecular docking simulations illuminated the interaction between ObGAPDH and hydrogen peroxide (H2O2) as a ligand. Scrutinizing the expression patterns of the ObGAPDH gene under conditions of water scarcity stress brought to light diverse levels of transcriptional activity. Collectively, these findings underscore the notion that the regulation of ObGAPDH expression is contingent upon both the specific plant cultivar and the presence of stress stemming from drought conditions.

Development and Evaluation of Recombinant B-Cell Multi-Epitopes of PDHA1 and GAPDH as Subunit Vaccines against Streptococcus iniae Infection in Flounder (Paralichthys olivaceus).

Streptococcus iniae is a severe Gram-positive pathogen that can infect a wide range of freshwater and marine fish species. In continuation of our earlier studies on the development of S. iniae vaccine candidates, pyruvate dehydrogenase E1 subunit alpha (PDHA1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were highly efficacious in protecting flounder (Paralichthys olivaceus) against S. iniae. In the present study, to investigate the potential of multi-epitope vaccination strategy to prevent flounder against S. iniae infection, the liner B-cell epitopes of PDHA1 and GAPDH proteins were predicted using a bioinformatics approach and were identified by immunoassay, and recombinant B-cell multi-epitopes of PDHA1 and GAPDH (rMEPIP and rMEPIG) containing immunodominant epitope-concentrated domains were expressed in Escherichia coli BL21 (DE3) and were used as a subunit vaccine to immunize healthy flounder, while recombinant PDHA1 (rPDHA1), GAPDH (rGAPDH) and formalin-inactivated S. iniae (FKC) served as controls. Then, the immunoprotection efficacy of rMEPIP and rMEPIG was evaluated by determining the percentages of CD4-1+, CD4-2+, CD8β+ T lymphocytes and surface-IgM-positive (sIgM+) lymphocytes in peripheral blood leucocytes (PBLs), spleen leucocytes (SPLs) and head kidney leucocytes (HKLs), as well as total IgM, specific IgM, and relative percentage survival (RPS) post immunization, respectively. It was found that fish immunized with rPDHA1, rGAPDH, rMEPIP, rMEPIG and FKC showed significant increases in sIgM+, CD4-1+, CD4-2+, and CD8β+ lymphocytes and production of total IgM and specific IgM against S. iniae or recombinant proteins rPDHA1 and rGAPDH, which indicated the activation of humoral and cellular immune responses after vaccination. Moreover, RPS rate of the multi-epitope vaccine rMEPIP and rMEPIG groups reached 74.07% and 77.78%, higher than that of rPDHA1 and rGAPDH (62.96% and 66.67%) and KFC (48.15%). These results demonstrated that B-cell multi-epitope protein vaccination, rMEPIP and rMEPIG, could give a better protective effect against S. iniae infection, which provided a promising strategy to design the efficient vaccine in teleost fish.

Loss of β-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1

Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.

A Comparison Between Recombinant Listeria GAPDH Proteins and GAPDH Encoding mRNA Conjugated to Lipids as Cross-Reactive Vaccines for Listeria, Mycobacterium, and Streptococcus.

Cross-reactive vaccines recognize common molecular patterns in pathogens and are able to confer broad spectrum protection against different infections. Antigens common to pathogenic bacteria that induce broad immune responses, such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the genera Listeria, Mycobacterium, or Streptococcus, whose sequences present more than 95% homology at the N-terminal GAPDH1−22 peptide, are putative candidates for universal vaccines. Here, we explore vaccine formulations based on dendritic cells (DC) loaded with two molecular forms of Listeria monocytogenes GAPDH (LM-GAPDH), such as mRNA carriers or recombinant proteins, and compare them with the same molecular forms of three other antigens used in experimental vaccines, listeriolysin O of Listeria monocytogeness, Ag85A of Mycobacterium marinum, and pneumolysin of Streptococcus pneumoniae. DC loaded with LM-GAPDH recombinant proteins proved to be the safest and most immunogenic vaccine vectors, followed by mRNA encoding LM-GAPDH conjugated to lipid carriers. In addition, macrophages lacked sufficient safety as vaccines for all LM-GAPDH molecular forms. The ability of DC loaded with LM-GAPDH recombinant proteins to induce non-specific DC activation explains their adjuvant potency and their capacity to trigger strong CD4+ and CD8+ T cell responses explains their high immunogenicity. Moreover, their capacity to confer protection in vaccinated mice against challenges with L. monocytogenes, M. marinum, or S. pneumoniae validated their efficiency as cross-reactive vaccines. Cross-protection appears to involve the induction of high percentages of GAPDH1−22 specific CD4+ and CD8+ T cells stained for intracellular IFN-γ, and significant levels of peptide-specific antibodies in vaccinated mice. We concluded that DC vaccines loaded with L. monocytogenes GAPDH recombinant proteins are cross-reactive vaccines that seem to be valuable tools in adult vaccination against Listeria, Mycobacterium, and Streptococcus taxonomic groups.

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein

BackgroundWe previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. MethodsHuman recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. ResultsS-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. ConclusionsS-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. General significanceThe described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

Organization of multi-binding to host proteins: The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a frequent cause of community-acquired infections of the human respiratory tract. During the evolutionary adaptation of the bacteria to the host, the genome of the pathogen is strongly reduced resulting in the loss of cell wall, limited metabolic pathways and a relatively small number of virulence factors. As interacting with host proteins, surface-exposed proteins with a primary function in cytosol-located processes of metabolism and regulation such as glycolytic enzymes, heat-shock proteins and chaperones have been considered as contributing to pathogenesis. Among these moonlighting proteins, some members are confirmed as binding to several host components. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of M. pneumoniae is a typical example of such multi-binding proteins. To investigate the organization of these interactions, GAPDH was divided into four parts. Recombinant proteins were successfully expressed in Escherichia coli and polyclonal antisera were produced. Binding of full length and parts of GAPDH to human A549 cells was proven. Furthermore, interactions with human plasminogen, vitronectin, fibronectin and fibrinogen were demonstrated for nearly all recombinant GAPDH proteins. In the presence of these proteins, plasminogen can be activated to the protease plasmin. In contrast, the localization on the surface of bacterial cell was confirmed for the C-terminal part of GAPDH only. By using overlapping peptides covering this region, binding of the investigated host components to the sequence 326QLVRVVNYCAKL337 was found. The results of the study suggest a prominent role of the surface-localized C-terminal part of GAPDH in associations with different human proteins indicating its importance for host-pathogen-interactions.

MAP1203 Promotes Mycobacterium avium Subspecies paratuberculosis Binding and Invasion to Bovine Epithelial Cells.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium.

Protective Efficacy of Coccidial Common Antigen Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) against Challenge with Three Eimeria Species.

Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several Eimeria species. GAPDH is one of the immunogenic common antigens among Eimeria tenella, E. acervulina, and E. maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of GAPDH cloned from E. acervulina and E. maxima were named as EaGAPDH and EmGAPDH, respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The EaGAPDH genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4+ and CD8+ T lymphocytes, the cytokines productions of IFN-γ, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species.

S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase in rodent muscle cells

S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by Western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independent of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

Lipid transfer protein CaMBP10 binds to glyceraldehyde-3-phosphate dehydrogenase and modulates its activity

Lipid transfer proteins (LTPs) are a protein family found in plants with a variety of functions. In addition to lipid binding, LTPs also bind to calmodulin and Ca2+-dependent protein kinase (CDPK), which are calcium signal transducers. For the first time, we identified glyceraldehyde-3- phosphate dehydrogenase (GAPDH) as a novel binding protein of LTP-CaMBP10 in Chinese cabbage. This binding was confirmed using multiple biochemical approaches. The effects of this interaction on GAPDH activity were assessed for both recombinant and endogenous GAPDH proteins. LTP-CaMBP10 does not appear to affect nicotinamide adenine dinucleotide (NAD)-dependent GAPDH activity. In contrast, it significantly suppresses nicotinamide adenine dinucleotide phosphate (NADPH) consumption by GAPDH in a dosage-dependent manner. This result indicated a specific role of GAPDH in regulating LTP functions and implicating crosstalk between LTP-dependent and GAPDH-dependent biological events.

Proteome profiling of the dimorphic fungusPenicillium marneffeiextracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment

Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicilliummarneffei remain largely unknown. By comparing the extracellular proteomes of P.marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P.marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P.marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P.marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P.marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional) properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively modified GAPDH in stress signaling pathways in plants.

Glyceraldehyde-3-phosphate dehydrogenase: A universal internal control for Western blots in prokaryotic and eukaryotic cells

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.

CP12 residues involved in the formation and regulation of the glyceraldehyde-3-phosphate dehydrogenase–CP12–phosphoribulokinase complex in Chlamydomonas reinhardtii

CP12, a member of the intrinsically disordered protein family, forms a stable complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). To understand the function of conserved residues of CP12 in the formation of the GAPDH-CP12-PRK complex and in the regulation of the enzymes within this complex, we have produced mutants of CP12 by site-directed mutagenesis. The GAPDH, CP12 and PRK recombinant proteins are able to reconstitute spontaneously the ternary complex that has been described in Chlamydomonas reinhardtii. Our analysis reveals that the central part ((35)WXXVEE(47)) of CP12 is required to form the GAPDH-CP12-PRK complex. Using the same series of single amino acid replacements, we have identified individual residues, which seem to represent also contact points for GAPDH. Most notably, substitution of glutamate 74 prevents the binding of GAPDH to CP12. This is similar to the mutant C66S, with which the GAPDH-CP12-PRK complex is not formed. In contrast, replacement of the three last residues ((78)YED(80)) of CP12 has no effect on the formation of the ternary supra-molecular complex. However, our findings strongly suggest that Y78 and D80 are involved in the regulation of the GAPDH activity within the supra-molecular complex, since the mutants, D80K and Y78S, do not down-regulate the activity of GAPDH. The replacement of the amino acid E79 weakens the interaction between GAPDH and CP12 as no GAPDH-CP12 sub-complex is formed. In this case, nevertheless, the supra-molecular complex is formed when PRK is present indicating that PRK strengthens the interaction between GAPDH and CP12 within the supra-molecular complex.

Edwardsiella tarda의 glyceraldehyde-3-phosphate dehydrogenase가 병원성에 미치는 영향

A research group demonstrated that the 37 kDA protein of Edwardsiella tarda, a causing causative agent of edwardsiellosis in fish, exhibited high antigenicity in Japanese flounder. The research group also showed that the N-terminus amino acid sequences of the 37 kDa protein were mapped to the N-terminus of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Using degenerated primer sets based on the known N-terminus sequence, the corresponding E. tarda DNA was amplified and cloned. The nucleotide sequences of the cloned gene revealed high homology with a bacterial gene for GAPDH, as we was expected. The amino acid sequence of E. tarda GAPDH (etGAPDH) revealed a <70% similarity with GAPDH proteins in other Enterobacteriaceae. With the application of artificial protein overexpression system in Escherichia coli, the recombinant etGAPDH (rGAPDH) was produced and purified. In this study, Using the purified rGAPDH, the etGAPDH specific polyclonal antibody has been was generated using the purified rGAPDHin this study. The immunoblotting analyses demonstrated that the location of the GAPDH protein is located with the association of is associated with the envelops of E. tarda. The rGAPDH was administrated into Japanese flounder via IP route for evaluation of the protective ability. Although the specific antibody titer against etGAPDH was increased about 3-fold after 4 weeks post-vaccination, the survival rates of vaccinated Japanese flounder and the control group with wild type E. tarda was were 12.5% and 0%, respectively. Our results indicated that rGAPDH is immunoreactive antigen but that it will not generate protective immunity in Japanese flounder.

Cloning and molecular characterization of glyceraldehyde-3-phosphate dehydrogenase gene from thermotolerant mushroom, Lentinus polychrous

This study describes the cloning and expression analysis of the heat shock protein gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in thermotolerant mushroom Lentinus polychrous, one of the most widely cultivated commercial edible mushroom in Thailand. The complete GAPDH coding sequence contained 1,011 bp, encoding for a polypeptide of 337 amino acid residues with the calculated molecular mass of 36.2 kDa and pI of 5.64. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the L. polychrous genome. The putative amino acid sequence of the L. polychrous GAPDH-encoded protein shared significant sequence identity with other GAPDH proteins from basidiomycetes. Phylogenetic analysis clustered the L. polychrous GAPDH protein with other homobasidiomycetes. Expression analysis of the GAPDH gene by RT-PCR showed that this gene was highly induced not only by heat, but also by cold, ethanol and salt stresses. The GAPDH gene was expressed in both mycelia and fruiting bodies, suggesting that the GAPDH gene product is a heat shock protein which might be involved in the developmental phase of the L. polychrous.

Structure of glyceraldehyde-3-phosphate dehydrogenase from the archaeal hyperthermophileMethanocaldococcus jannaschii

The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 A resolution. The crystal belonged to space group C222(1), with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 A. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R(free) = 19.8%). The final structure included the cofactor NADP(+) at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal alpha-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site.