Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Diabetic plasma contains elevated levels of glucose and various low-molecular-weight carbonyl compounds derived from the metabolism of glucose and related materials. These compounds react with protein side chains (Arg, Lys, Cys, and His) to give glycated materials and advanced glycation end products. In this study, we have examined the effect of glucose and carbonyl compounds (methylglyoxal, glyoxal, glycolaldehyde, and hydroxyacetone), and glycation products arising from reaction of these materials with model proteins, on the activity of three key cellular enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione reductase, and lactate dehydrogenase, both in isolation and in cell lysates. In contrast to glucose (1M, both fresh and aged for 8 weeks), which had no effect, marked inhibition of all three enzymes was observed with methylglyoxal and glyoxal. GAPDH was also inhibited by glycolaldehyde and hydroxyacetone. Incubation of these enzymes with proteins that had been preglycated with methylglyoxal, but not glucose, also resulted in significant time- and concentration-dependent inhibition with both isolated enzymes and cell lysates. This inhibition was not metal ion, oxygen, superoxide dismutase, or catalase dependent, suggesting that inhibition is not radical mediated. These effects are suggested to be due to direct adduction of the free- or protein-bound carbonyls with the target enzyme. Such an interpretation is supported by the detection of the loss of thiol groups on GAPDH and the detection of cross-linked materials on protein gels. Though direct comparison of the extent of inhibition induced by free versus protein-bound carbonyls was not possible, the significantly higher concentrations of the latter materials over the former in diabetic plasma and cells lead us to suggest that alterations in the activity of key cellular enzymes induced by glycated proteins may play a significant role in the development of diabetic complications.
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PMID:Inactivation of cellular enzymes by carbonyls and protein-bound glycation/glycoxidation products. 1213 75

Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a K(d) of 70 +/- 11 nM and 112 +/- 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by epsilon ACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C-terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen-binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C-terminal lysine residues and three (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface-bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.
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PMID:Candida albicans binds human plasminogen: identification of eight plasminogen-binding proteins. 1262 18

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.
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PMID:Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase. 1265 51

Although the transplantation of alginate-poly-L-lysine-alginate encapsulated islets of Langerhans usually is successful, graft survival is still limited. Molecular analysis by RT-PCR of the encapsulated islets may provide insight into the mechanisms that affect islets during graft failure. However, RT-PCR on encapsulated islets is not possible because the poly-L-lysine of the capsule interferes with both cDNA synthesis and PCR amplification. We applied a method that mechanically removes the microcapsules from the islets after a short trypsin-EDTA treatment (decapsulation), thereby enabling RT-PCR analysis. The results of this study show that the decapsulation procedure does not affect islet vitality and has only minor effects on islet function and morphology. The decapsulation does not affect GAPDH, beta-actin, Bcl-2, or Bax gene expression. This method is an improvement over the time-consuming manual dissection of microcapsules because it allows for the rapid and relatively harmless removal of capsules on a larger scale. Decapsulation offers the possibility of applying RT-PCR, as well as other methods, which cannot be performed on encapsulated islets.
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PMID:Effective removal of alginate-poly-L-lysine microcapsules from pancreatic islets by use of trypsin-EDTA. 1456 13

Histone modifications have emerged to be a major regulatory mechanism for gene expression (1-4). However, it is not clear how histone modifications are physiologically regulated. Here, we show that mono-ubiquitinated H2B at lysine 123 (uH2B) in the yeast (Saccharomyces cerevisiae) is present in exponential phase and absent in stationary phase. A wide array of carbohydrates or sugars, including glucose, fructose, mannose, and sucrose, are capable of inducing uH2B in stationary phase yeast. In contrast, non-metabolic glucose analogs are defective in inducing uH2B. Furthermore, uH2B induction is inhibited by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase in glycolysis. Moreover, uH2B induction is markedly impaired in yeast mutants, in which glycolytic genes are deleted. These data indicate that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B at lysine 123. Therefore, our study reveals a novel paradigm of metabolic regulation of histone modifications.
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PMID:Carbohydrates induce mono-ubiquitination of H2B in yeast. 1466 Jun 35

Brownian dynamics simulations of computer models of GAPDH mutants interacting with F-actin emphasized the electrostatic nature of such interactions, and confirmed the importance of four previously identified lysine residues on the GAPDH structure in these interactions. Mutants were GAPDH models in which one or more of the previously identified lysines had been replaced with alanine. Simulations showed reduced binding of these mutants to F-actin compared to wild-type GAPDH. Binding was significantly reduced by mutating the four lysines; the specific electrostatic interaction energy of the quadruple mutant was -7.3 +/- 1.0 compared to -11.4 +/- 0.5 kcal/mol for the wild enzyme. The BD simulations also reaffirmed the importance of quaternary structure for GAPDH binding F-actin.
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PMID:Brownian dynamics of interactions between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mutants and F-actin. 1504 77

Many early pregnancy complications are associated with an imbalance in pro- and anti-inflammatory cytokines, resulting in alterations in nitric oxide (NO) profile. Since very little is known about the modus operandi of this free radical in early embryos, this study characterised NO embryotoxicity in terms of bovine embryo development and metabolism. Embryos were generated by in vitro maturation and fertilisation of oocytes aspirated from abattoir-derived ovaries. Zygote to blastocyst rates were measured in SOFaaBSA in the presence and absence of the NO donor sodium nitroprusside (SNP) over the 0-50 microM range (n=10 per group). Since concentrations <10 microM SNP depressed blastocyst rate, blastocyst cell numbers (determined by bisbenzimide staining; n=22 and 20), glucose, pyruvate, lactate (measured ultramicrofluorometrically) and amino acid profiles (quantified by HPLC; n=28 and 23) were assessed at 0 and 10 microM SNP. SNP depressed cell numbers, reduced pyruvate and glucose uptake, perturbed quantitative tyrosine, threonine, phenylalanine, lysine, glycine, tryptophan, methionine and valine profiles, and decreased retention into the negative range (P<0.05). Qualitative asparagine and lysine profiles were affected by SNP, while proportional amino acid production and consumption were increased and decreased, respectively (P<0.05). These findings indicate that SNP (presumably through increases in NO profile): (i) fails to improve bovine embryo development in vitro, (ii) exerts toxic effects, likely through ATP starvation induced by cytochrome c oxidase (oxidative phosphorylation) and glyceraldehyde-3-phosphate dehydrogenase (glycolysis) inhibition, and (iii) may affect albumin endocytosis/hydrolysis or protein biosynthesis, rather than causing a loss of intracellular amino acids or simply depressing their metabolism.
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PMID:Embryotoxicity of the nitric oxide donor sodium nitroprusside in preimplantation bovine embryos in vitro. 1596 59

Although biologically active, nitroxyl (HNO) remains one of the most poorly studied NO(x). Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addition product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 microM), human calbindin D(28k) (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivatives in incubates containing 0.9-2.5 mM AS. Ions at M + n x 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramolecular sulfinamide cross-linking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were observed for HCalB, which additionally formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramolecular disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3(-) --> HNO + NO2(-)), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.
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PMID:Mass spectrometric analysis of nitroxyl-mediated protein modification: comparison of products formed with free and protein-based cysteines. 1622 92

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.
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PMID:S-(2-Succinyl)cysteine: a novel chemical modification of tissue proteins by a Krebs cycle intermediate. 1662 47

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.
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PMID:Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols. 1667 91


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