Mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in the presence of methylglyoxal.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be one of the targets of methylglyoxal (MGO), a metabolite of glycolysis that increased in diabetes. However, the mechanism of GAPDH inactivation in the presence of MGO is unclear. The purpose of the work was to study the reaction of GAPDH with MGO and to identify the products of the reaction. It was shown that incubation of recombinant human GAPDH with MGO leads to irreversible inactivation of the enzyme, which is accompanied by a decrease in SH-group content by approximately 3.3 per tetramer GAPDH. MALDI-TOF MS analysis showed that the modification of GAPDH with MGO results in the oxidation of the catalytic cysteine residues (Cys152) to form cysteine-sulfinic acid. In addition, 2 arginine residues (R80 and R234) were identified that react with MGO to form hydroimidazolones. Incubation of SH-SY5Y neuroblastoma cells with MGO resulted in the inactivation of GAPDH and inhibition of glycolysis. The mechanism of GAPDH oxidation in the presence of MGO suggests the participation of superoxide anion, which is formed during the reaction of amino groups with methylglyoxal. The role of GAPDH in protection against the damaging effect of ROS in cells in the case of inefficiency of MGO removal by the GSH-dependent glyoxalase system is discussed.

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

BACKGROUND: We 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. METHODS: Human 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. RESULTS: S-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. CONCLUSIONS: S-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 SIGNIFICANCE: The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

Inhibitors of Glyceraldehyde 3-Phosphate Dehydrogenase and Unexpected Effects of Its Reduced Activity.

The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. METHODS: Rabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. RESULTS: Incubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD+ and thermal stability. CONCLUSIONS: The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. GENERAL SIGNIFICANCE: The manuscript considers one of the possible mechanisms of redox regulation of cell functions.

Isolation of recombinant human untagged glyceraldehyde-3-phosphate dehydrogenase from E. coli producer strain.

The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass). The specific activity of the freshly purified hGAPDH constitutes 117 ± 5 µmol NADH/min per mg protein (pH 9.0, 22 °C), which is close to the specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase determined under the same conditions and several times exceeds the specific activity of his-tagged GAPDH preparations. The high enzymatic activity suggests that the recombinant enzyme retains its native structure. The described procedure may be useful for researchers who need a preparation of native hGAPDH without admixture of misfolded forms for their investigations.

Dimerization of Tyr136Cys alpha-synuclein prevents amyloid transformation of wild type alpha-synuclein.

Expression of human alpha-synuclein in E. coli cells is known to result in a mixture of the wild type alpha-synuclein and the protein containing Tyr136Cys substitution due to the translational error. The amount of Cys136 alpha-synuclein (Cys136-AS) may reach approximately 50% of the recombinant protein. The wild-type and Cys136-containing fractions of alpha-synuclein were separated using thiol-Sepharose, and their properties were investigated. In the absence of reducing agents, Cys136-AS forms dimers due to the disulfide bonding. Both wild-type and Cys136 alpha-synuclein preparations are prone to aggregate during prolonged incubation under shaking at pH 4 and 37°C, but only the wild-type alpha-synuclein produces amyloid aggregates. The aggregates produced by either monomeric or dimeric Cys136-AS do not exhibit amyloid properties according to the test with Thioflavin T. Moreover, an admixture of dimeric Cys136-AS prevents the amyloid transformation of the wild-type alpha-synuclein. CD spectroscopy analysis revealed an enhanced content of alpha-helical structures in the aggregates produced by dimeric Cys136-AS. The admixture of Cys136-AS in preparations of human recombinant alpha-synuclein can be a source of erroneous interpretation of experiments on amyloid transformation of this protein.

Low Concentrations of Hydrogen Peroxide Activate the Antioxidant Defense System in Human Sperm Cells.

The effect of low concentrations of hydrogen peroxide (10-100 µM) on sperm motility and on the activity of the sperm enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) was investigated. Incubation of semen samples with 10 and 100 µM hydrogen peroxide increased the content of spermatozoa with progressive motility by 20 and 18%, respectively, and enhanced the activity of GAPDS in the sperm cells by 27 and 20% compared to a semen sample incubated without additions. It was also found that incubation with 10 µM hydrogen peroxide increased the content of reduced glutathione (GSH) in sperm cells by 50% on average compared to that in the control samples. It is supposed that low concentrations of hydrogen peroxide activate the pentose phosphate pathway, resulting in NADPH synthesis and the reduction of the oxidized glutathione by glutathione reductase yielding GSH. The formed GSH reduces the oxidized cysteine residues of the GAPDS active site, increasing the activity of the enzyme, which in turn enhances the content of sperm cells with progressive motility. Thus, the increase in motile spermatozoa in the presence of low concentrations of hydrogen peroxide can serve as an indicator of normal functioning of the antioxidant defense system in sperm cells.

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.