Role Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Differential gene expression and dependence on thyroid hormones of two glyceraldehyde-3-phosphate dehydrogenases in the flatfish Senegalese sole ( Solea senegalensis Kaup)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme of the glycolytic pathway. The application of large-scale genomics to Senegalese sole ( Solea senegalensis) has facilitated the identification of two different genes referred to as GAPDH-1 and GAPDH-2. Main characteristics and sequence similarities with other fish and mammals are described. Phylogenetic analyses grouped both genes into two separate clusters with their mammalian counterparts as a sister clade. The expression profiles of both genes were studied during larval development and in juvenile tissues using a real-time PCR approach. In juvenile fish, GAPDH-1 was expressed mainly in muscle and GAPDH-2 in brain. During metamorphosis, GAPDH-2 mRNA levels did not change. In contrast, GAPDH-1 transcripts increased significantly between S1 and S2 metamorphic stages and reduced their levels thereafter. A treatment with the goitrogen thiourea indicated that GAPDH-1 expression was negatively regulated by thyroid hormones. The possible role of GAPDHs in metamorphosis is discussed.

Forward chemical genetic approach identifies new role for GAPDH in insulin signaling

Insulin and insulin-like growth factor have an essential role in growth, development and the maintenance of metabolic homeostasis, including glucose uptake from the bloodstream. Researchers have identified mutations in insulin receptors that cause severe insulin resistance, and a temperature-sensitive daf-2 (a gene encoding an insulin receptor-like protein) mutant in Caenorhabditis elegans has served as an insulin resistance model. Here we report a forward chemical genetic approach with a tagged library that we used to identify a small molecule, GAPDH segregator (GAPDS), that suppresses the dauer formation induced by the daf-2 mutant. Like insulin, GAPDS increased both glucose uptake and the concentration of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in mammalian preadipocytes. Using affinity matrices and RNA interference, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a GAPDS target. We discovered that GAPDH stimulates phosphatase activity against not only PtdIns(3,4,5)P(3) but also PtdIns(4,5)P(2). These results suggest that GAPDH is both an active regulator in the phosphoinositide-mediated signaling pathway and a potential new target for insulin resistance treatment.

Bacterial endotoxin lipopolysaccharide induces up-regulation of glyceraldehyde-3-phosphate dehydrogenase in rat liver and lungs

Bacterial endotoxin or lipopolysaccharide (LPS) can trigger inflammatory responses and cause damage in organs such as liver and lungs when it is introduced into mammals, but the exact molecular events that mediate these responses have remained obscure. In this study, by using 2D gel electrophoresis and cDNA microarray analysis, we found that both protein and mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were significantly increased in rat liver and lungs after treatment with LPS. The results were further confirmed by Western blot and Northern blot. Given the known role of GAPDH in inducing apoptosis, our results suggest that LPS-induced GAPDH up-regulation may be an important mechanism responsible for the damage induced by Gram negative bacteria in mammalian tissue and GAPDH may be involved in the signaling pathway of LPS induced apoptosis. Our results also demonstrate that GAPDH is not a suitable internal control in gene expression studies, especially when bacterial infection is involved.

Association of late-onset Alzheimer's disease with genetic variation in multiple members of the GAPD gene family

Although several genes have been implicated in the development of the early-onset autosomal dominant form of Alzheimer's disease (AD), the genetics of late-onset AD (LOAD) is complex. Loci on several chromosomes have been linked to the disease, but so far only the apolipoprotein E gene has been consistently shown to be a risk factor. We have performed a large-scale single-nucleotide polymorphism (SNP)-based association study, across the region of linkage on chromosome 12, in multiple case-control series totaling 1,089 LOAD patients and 1,196 control subjects and report association with SNPs in the glyceraldehyde-3-phosphate dehydrogenase (GAPD) gene. Subsequent analysis of GAPD paralogs on other chromosomes demonstrated association with two other paralogs. A significant association between LOAD and a compound genotype of the three GAPD genes was observed in all three sample sets. Individually, these SNPs make differential contributions to disease risk in each of the casecontrol series, suggesting that variants in functionally similar genes may account for series-to-series heterogeneity of disease risk. Our observations raise the possibility that GAPD genes are AD risk factors, a hypothesis that is consistent with the role of GAPD in neuronal apoptosis.

Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin.

Mycoplasma genitalium is the smallest known self-replicating cell. It was first isolated from urethral specimens in individuals with non-gonococcal urethritis and, more recently, from respiratory and synovial sites. Our laboratory has been interested in defining the mechanisms by which M. genitalium adheres to and colonizes host cell surfaces. In order to determine potential targets of adherence, we examined the interaction of M. genitalium with a primary component of the mucosal epithelial lining, mucin (Mn). Three Mn-binding proteins (MnBPs) of M. genitalium were isolated by affinity chromatography. One of these proteins was identified by N-terminal sequencing as glyceraldehyde-3-phosphate dehy-drogenase (GAPDH). Antiserum raised against recombinant GAPDH blocked binding of intact biosynthetically labelled mycoplasmas to mucin by approximately 70%. Whole cell radioimmunoprecipitation indicated that GAPDH was surface-accessible and surface localization of GAPDH was further verified by membrane fractionation and immunoelectron microscopy. The role of GAPDH as an adhesin to Mn not only provides insights into the organism's mechanisms of adherence and colonization but also into its ability to maximize its limited genome.

Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress.

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H(2)O(2) or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.

A proteomic analysis of erythromycin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant human pathogen which is an important cause of pneumonia and bacteraemia. Over the past few years the incidence of antibiotic resistance among clinical isolates of S. pneumoniae has increased. Penicillin resistance is now widespread and the frequency of isolates that are resistant to erythromycin has risen. Erythromycin resistance in S. pneumoniae follows two basic patterns. The MLS erythromycin-resistant phenotype is due to the enzymatic methylation of ribosomal RNA that blocks erythromycin binding to the ribosome. Alternatively, in isolates of the M phenotype, a more recently documented mechanism, resistance is associated with an active efflux process that reduces intracellular levels of erythromycin. We used two-dimensional electrophoresis to examine the proteins synthesised by erythromycin-susceptible and -resistant S. pneumoniae. Erythromycin-resistant S. pneumoniae with the M phenotype showed a significantly increased synthesis of a 38,500 Dalton (pI 6.27) protein compared to susceptible isolates. Peptide mass mapping was used to identify the 38,500 Dalton protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was demonstrated that S. pneumoniae synthesised at least three forms of GAPDH that differed in their isoelectric points. The form of GAPDH possessing the most basic pI showed the increased synthesis in the erythromycin-resistant S. pneumoniae isolates. Alterations in the synthesis of GAPDH were only found for those erythromycin-resistant isolates possessing the M phenotype. S. pneumoniae isolates with the MLS phenotype were indistinguishable from the susceptible strains using the analytical conditions employed for the current study. The possible role of GAPDH in erythromycin resistance of S. pneumoniae is considered.

Increased expression of glyceraldehyde-3-phosphate dehydrogenase in cultured astrocytes following exposure to manganese

Manganism is a disorder characterized by hyperintensities in basal ganglia structures on magnetic resonance imaging which may be the consequence of manganese deposition in these areas. Since manganese is taken up avidly into astrocytes and is known to interfere with cerebral energy metabolism, we studied the effect of this metal on the expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in primary cultures of astrocytes. Treatment with 100 μM manganese for 7 days increased both the V max and K m values for GAPDH which was not reproducible with other divalent metals. Using RT-PCR, increased GAPDH expression was detected in cells exposed to manganese compared with controls. No changes in cytochrome oxidase activity or ATP levels were observed, and lactate production was unaffected, in manganese-treated cells. These findings provide evidence of a possible role for GAPDH in the mediation of the effects of manganese on central nervous system function.

Disappearance of glyceraldehyde 3-phosphate dehydrogenase from erythrocyte membrane by hemolysis with thermostable direct hemolysin of Vibrio parahaemolyticus or Vibrio cholerae El Tor hemolysin.

It is believed that the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus and El Tor hemolysin (ETH) of Vibrio cholerae damage erythrocytes and other cells by acting as pore-forming toxins. In this study, we found that a protein band with a molecular weight of 37,000 daltons specifically disappeared from erythrocyte membrane after hemolysis by TDH and ETH, but not after hypotonic hemolysis. The 37 kDa band was identified as glyceraldehyde 3-phosphate dehydrogenase (G3PD), a glycolytic enzyme, based on N-terminal 14 amino acid sequencing. The role of G3PD in hemolysis is discussed.

The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide.

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.

Metabolic control mechanisms in human erythrocytes the role of glyceraldehyde phosphate dehydrogenase

Hemolyzates of human red cells have been utilized to evaluate the role of glyceraldehyde 3-phosphate dehydrogenase as a control enzyme in the glycolytic pathway. The kinetic data obtained at pH 7.3 indicate Km values of 21 μ m , 143 μ m , and 6.6m m , respectively, for glyceraldehyde 3-phosphate, NAD, and inorganic phosphate. NADH is a competitive inhibitor of NAD with a Ki of 9.1 μ m . At physiologic concentrations of substrates and NADH, glyceraldehyde phosphate dehydrogenase is operating at less than 1% of maximal velocity. Consequently, despite the relatively high activity reported for this enzyme in erythrocytes, it appears likely that it is a site of metabolic control. It is clearly the rate-limiting reaction when the pH of the cells is raised or when the phosphate concentration of the external medium is increased markedly.