GAPDH binders as potential drugs for the therapy of polyglutamine diseases: Design of a new screening assay

AbstractProteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and −(−)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution

<p>Abstract</p> <p>Background</p> <p>Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein.</p> <p>Results</p> <p>A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences.</p> <p>Conclusions</p> <p>The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.</p

Polyelectrolytes for Enzyme Immobilization and the Regulation of Their Properties

In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems (self-regulated enzyme&ndash;polyelectrolyte complexes, artificial chaperones, polyelectrolyte brushes, layer-by-layer immobilization and others). These works represent the field of &ldquo;smart polymers&rdquo;, whilst the trivial use of charged polymers as carriers for adsorption or covalent immobilization of proteins is beyond the scope of this short review. In addition, we have included a section on the molecular modeling of interactions between proteins and polyelectrolytes, as modeling the binding of proteins with a strictly defined, and already known, spatial structure, to disordered polymeric molecules has its own unique characteristics

Sorted Bulls’ X-Chromosome-Bearing Spermatozoa Show Increased GAPDHS Activity Correlating with Motility

Sperm sexing is a technique for spermatozoa sorting into populations enriched with X- or Y-chromosome-bearing cells and is widely used in the dairy industry. Investigation of the characteristics of sorted semen is of practical interest, because it could contribute to the enhancement of sexed semen fertility characteristics, which are currently lower than those of conventional semen. Comparison of a spermatozoa population enriched with X-chromosome-bearing cells to a mixed population is also intriguing in the context of potential differences that drive the mechanisms of primary sex-ratio determination. In this work, sexed (X spermatozoa) and conventional spermatozoa of Holstein bulls were analyzed for the content and enzymatic activity of GAPDHS, a sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase that plays a significant role in the regulation of flagellar activity. No difference in the amount of this glycolysis enzyme per cell was revealed, but, notably, GAPDHS enzymatic activity in the sexed samples was significantly higher. Enzymatic activity among the group of sexed but not conventional sperm samples positively correlated with spermatozoa motility, which indicates the significant role of this enzyme for the sorted cells population

Influence of complexing polyanions on the thermostability of basic protiens.

Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water-soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N-ethyl-4-vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion-protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein-polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo

Interaction of glyceraldehyde-3-phosphate dehydrogenase with SH-containing compounds: evidence for the binding of l-cysteine and for the dependence of the binding on the functional state of the enzyme

AbstractIncorporation of l-[35S]cysteine into rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde-3-phosphate, NAD+ and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of l[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The l-[35S]cysteine incorporation depended on pH and was maximal when a group having pKa of 8.5 is protonated. To clarify the relevance of this finding to the effect of SH-containing compounds previously shown to decrease the rate of 3-phosphoglyceroyl-enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208–210], the pH-dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH-dependence of l-[35S]cysteine binding

Influence of Complexing Polyanions on the Thermostability of Basic Proteins

Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water-soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N-ethyl-4-vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion-protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein-polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo

Differential Analysis of A-to-I mRNA Edited Sites in Parkinson&rsquo;s Disease

Parkinson&rsquo;s disease (PD) is a widespread neuronal degenerative disorder with unexplored etiology. It is associated with various pathological events. In particular, the prefrontal cortex Brodmann area 9 (BA9) region is affected in PD. This frontal lobe brain region plays an important role in cognitive, motor, and memory-related functions. BA9 develops Lewy bodies in PD patients and shows essential changes in transcriptome and proteome, connected with mitochondria related pathways, protein folding pathways, and metallothioneins. Recently, altered adenosine to inosine mRNA editing patterns have been detected in various neurological pathologies. In this article, we present an investigation of differences in A-to-I RNA editing levels and specificity of mRNA editing sites in brain tissues of healthy and PD patients based on RNA sequencing data. Overall, decreased editing levels in the brains of PD patients were observed, potential editing sites with altered editing during PD were identified, and the role of different adenosine deaminases in this process was analyzed

Influence of oxidative stress on catalytic and Non-glycolytic Functions of Glyceraldehyde-3-phosphate Dehydrogenase

BACKGROUND: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) is a unique enzyme that, besides its main function in glycolysis (catalysis of glyceraldehyde-3-phosphate oxidation), possesses a number of non-glycolytic activities. The present review summarizes information on the role of oxidative stress in the regulation of the enzymatic activity as well as non-glycolytic functions of GAPDH. METHODS: Based on the analysis of literature data and the results obtained in our research group, mechanisms of the regulation of GAPDH functions through the oxidation of the sulfhydryl groups in the active site of the enzyme have been suggested. RESULTS: Mechanism of GAPDH oxidation includes consecutive oxidation of the catalytic Cysteine (Cys150) into sulfenic, sulfinic, and sulfonic acid derivatives, resulting in the complete inactivation of the enzyme. The cysteine sulfenic acid reacts with reduced glutathione (GSH) to form a mixed disulfide (S-glutathionylated GAPDH) that further reacts with Cys154 yielding the disulfide bond in the active site of the enzyme. In contrast to the sulfinic and sulfonic acids, the mixed disulfide and the intramolecular disulfide bond are reversible oxidation products that can be reduced in the presence of GSH or thioredoxin. CONCLUSION: Oxidation of sulfhydryl groups in the active site of GAPDH is unavoidable due to the enhanced reactivity of Cys150. The irreversible oxidation of Cys150 is prevented by Sglutathionylation and disulfide bonding with Cys154. The oxidation/reduction of the sulfhydryl groups in the active site of GAPDH can be used for regulation of glycolysis and numerous side activities of this enzyme including the induction of apoptosis