A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos

During heat shock, Xenopus laevis embryos exhibit an increase in the rate of accumulation of lactate and a loss of ATP relative to non-heat- shocked control embryos. These results suggest that heat shock stimulates a shift in energy metabolism to anaerobic glycolysis while at the same time causing an increase in the demand for ATP. We have evidence indicating that the embryo may meet such demands placed on it by increasing the levels of some glycolytic enzymes. In this report, we show that heat shock stimulates increases in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [( EC 1.2.1.12] GAPDH). The specific activity of GAPDH shows a significant increase after heat shock, which correlates with the accumulation of GAPDH in heat-shocked embryos as detected by immunoblotting. Increases in GAPDH-specific activity are variable, however, and are inversely proportional to the levels of specific activity in control embryos; i.e., constitutive enzyme activity. We further analyzed the heat-enhanced accumulation of GAPDH by electrophoretically separating GAPDH isozymes on nondenaturing polyacrylamide gels. Control embryos exhibit a single isozyme of GAPDH, whereas heat-shocked embryos exhibit two isozymes of GAPDH. When these isozymes are labeled with [35S]methionine, separated by nondenaturing gel electrophoresis, and analyzed by fluorography, a heat-shock protein is found to comigrate with the isozyme unique to the heat-shocked sample. Enzyme activity assays at different temperatures suggest that this isozyme has optimum enzymatic activity only at heat-shock temperatures. We have correlated a 35-kD heat-shock protein (hsp35) with GAPDH using the following evidence: this hsp comigrates with GAPDH on one-dimensional SDS polyacrylamide gels; heat-enhanced increases in GAPDH specific activity correlate with hsp35 synthesis; and hsp35 and GAPDH have similar peptide maps. This relationship also provides a compelling explanation for the restriction of hsp35 synthesis to the vegetal hemisphere cells of heat-shocked early gastrulae reported previously (Nickells, R. W., and L. W. Browder. 1985. Dev. Biol. 112:391-395)

Circadian Rhythm in Yeast

Circadian rhythms are found in a variety of organisms and are involved in controlling activity of the organism. However, a circadian rhythm for yeast has not been discovered yet. The goal of this study is to find evidence for a circadian rhythm in yeast by looking for changes in GAPDH concentrations and peroxiredoxin levels. GAPDH and peroxiredoxin have been shown to be controlled by the circadian clock in many organisms. A continuous yeast culture is sampled every two hours over a period of about 104 hours. Each sample is processed and tested for the concentration of GAPDH using GAPDH reagent. Western blots will also be performed to determine peroxiredoxin levels

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. © 2011 The Author(s)

Down-regulation of serotonergic genes expression in the raphe nuclei of midbrain under chronic social defeat stress in male mice

Background: 
There is ample experimental evidence supporting the hypothesis that the brain serotonergic system is involved in the control of chronic social defeat stress (CSDS), depression and anxiety. The study aimed to analyze mRNA levels of the serotonergic genes in the raphe nuclei of the midbrain that may be associated with chronic social defeats consistently shown by male mice in special experimental settings. 

Methodology/Principal Findings: 
The serotonergic genes were the Tph2, Sert, Maoa and Htr1a. The Bdnf, Creb, Cphn, Gapdh, Hprt, B2M, 18S and Actb genes were also studied. The experimental groups were composed of male mice with experience of defeats in 21 daily encounters and male mice with the same track record of defeats followed by a no-defeat period without agonistic interactions (relative rest for 14 days). It has been shown that mRNA levels of the Tph2, Maoa, Sert, Htr1a, Bdnf and Creb genes in the raphe nuclei of defeated mice are decreased as compared with the controls. Under CSDS the Cphn, Gapdh, Hprt, B2M, 18S, Actb genes are also down-regulated. The expression of the serotonergic genes as well as the Cphn and Creb genes is not restored to the control level after the 2 weeks of relative rest. mRNA levels of other genes are not recovered to the control levels, although some up-regulation was observed in rested losers. Significant positive correlations were found between the total time of avoidance behavior demonstrated by the 21-day defeaters in agonistic interactions and Sert, Maoa, Bdnf, Gapdh and 18S mRNA levels. 

Conclusions: 
CSDS experience inducing the development of mixed anxious/depression-like state in male mice down-regulates the serotonergic genes expression associated with the synthesis, inactivation and reception of serotonin. The Bdnf and Creb genes as well as the cell and metabolic Cphn, Gapdh, Hprt, B2M, Actb and 18S genes in the midbrain raphe nuclei are also down-regulated under CSDS. Period of relative rest is not enough for most genes to recover expression to the control levels

Disruption of the nuclear p53-GAPDH complex protects against ischemia-induced neuronal damage

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is conventionally considered a critical enzyme that involves in glycolysis for energy production. Recent previous studies have suggested that GAPDH is important in glutamate-induced neuronal excitotoxicity, while accumulated evidence also demonstrated that GAPDH nuclear translocation plays a critical role in cell death. However, the molecular mechanisms underlying this process remain largely unknown. In this study, we showed that GAPDH translocates to the nucleus in a Siah1-dependent manner upon glutamate stimulation. The nuclear GAPDH forms a protein complex with p53 and enhances p53 expression and phosphorylation. Disruption of the GAPDH-p53 interaction with an interfering peptide blocks glutamate-induced cell death and GAPDH-mediated up-regulation of p53 expression and phosphorylation. Furthermore, administration of the interfering peptide in vivo protects against ischemia-induced cell death in rats subjected to tMCAo. Our data suggest that the nuclear p53-GAPDH complex is important in regulating glutamate-mediated neuronal death and could serve as a potential therapeutic target for ischemic stroke treatment

Highly Mutable Linker Regions Regulate HIV-1 Rev Function and Stability.

HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev's established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev's ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein

NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study, enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains were shown to secrete GAPDH and the protein to bind human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification, which involves Cys-149 at the active site. ADP-ribosylation of extracellular GAPDH may play an important role in the host-pathogen interaction, as also proposed in other pathogens. [Int Microbiol 2009; 12(3):187-192

Glyceraldehyde-3-phosphate dehydrogenase as a moonlighting protein in bacteria

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/63704Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping protein that is present in virtually all organisms, where it performs metabolic functions essential for survival. GAPDH plays an essential role in the process of energy production, and is also involved in numerous biological processes. GAPDH belongs to a subset of proteins called moonlighting proteins, in which different functions are associated with a single polypeptide chain. The multifunctionality of GAPDH has been described in pathogenic and probiotic microorganisms, in mammals and in plants. In this review, we summarize the moonlighting role of GAPDH in bacteria