Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

A Major Outer Membrane Protein of Rahnella aquatilis Functions as a Porin and Root Adhesin

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60°C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand β-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.     

Preparation and molecular characterization of a polyclonal antibody as an efficient cutworm reference protein

Antibodies, which are wildly used in molecular researches, are proteins secreted by plasma cells that can specifically recognize specific antigens. To obtain a stable reference antibodies are important for protein analysis. In this study, a capable glyceraldehyde phosphate dehydrogenase (GAPDH) antiserum against most noctuid larval protein samples was prepared. Firstly, the full-length gapdh was amplified from Spodoptera exigua larvae to construct the prokaryotic expression vector. The purified His-tag fused GAPDH protein was used to immunize rabbits for the antiserum preparation. Protein samples extracted from 11 insect species distributed across seven families and three orders were used to perform the Western bolt analysis. The bright specific bands were detected in S. exigua, S. litura, Helicoverpa armigera, and Mythimna seperata protein samples, indicating that this antiserum may be capable of detecting noctuid larval encoded GAPDH. Further immunofluorescence identification of H. armigera and S. exigua paraffin section slides with GAPDH antiserum exhibited an identical cyto-stained image. The GAPDH antiserum prepared in this study is useful and efficient as reference antibodies in studies involving noctuid larval protein samples in other related fields.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

Here we describe the interaction of phosphorylated ∼40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Structured summary

MINT-7891324, MINT-7891304, MINT-7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)     

Isolation and Characterization of Vibrio tubiashii Outer Membrane Proteins and Determination of a toxR Homolog

Outer membrane proteins (OMPs) expressed by Vibrio tubiashii under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and ompU gene, a maltoporin-like protein, several novel OMPs, and a regulatory toxR homolog.     

Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.     

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.     

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.     

Subcellular Distribution of Glyceraldehyde-3-Phosphate Dehydrogenase in Cerebellar Granule Cells Undergoing Cytosine Arabinoside-Induced Apoptosis

Abstract: We have previously shown that cytosine arabinoside (AraC)-induced apoptosis of cerebellar granule cells (CGCs) results in an increase of a 38-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Antisense oligonucleotides to GAPDH mRNA afford acutely plated CGCs significant protection against AraC-induced apoptosis. We used differential centrifugation to examine which subcellular components are affected. Treated and untreated cells were sonicated in 0.32 M sucrose and sequentially centrifuged at 1,000, 20,000, and 200,000 g , to obtain crude nuclear, mitochondrial, microsomal, and cytosolic fractions. Western blotting showed that the levels of GAPDH protein were markedly increased in the 1,000- and 20,000- g pellets. The levels in the cytosolic supernatant were decreased dramatically by AraC in acutely plated CGCs but not in cells 24 h after plating. It is noteworthy that although GAPDH protein in the pellet fractions increased, the dehydrogenase activity of GAPDH decreased. Two other dehydrogenases, lactate dehydrogenase (EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), were not similarly affected, suggesting that the effect was GAPDH specific. These observations suggest that GAPDH levels change in specific organelles during apoptosis for reasons that are separate from its function as a glycolytic enzyme. The accumulation of GAPDH protein in specific subcellular loci may play a role in neuronal apoptosis.     

Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities

Hermetical microwave was used to degrade Porphyridium cruentum polysaccharides from 2918 to 256.2, 60.66 and 6.55 kDa. The antioxidant properties of different molecular weight polysaccharides were evaluated by determining the scavenging ability of free radicals, inhibitory effects on lipid peroxidation in liver homogenates and hemolysis of mouse erythrocytes. Analysis of physicochemical properties confirmed that microwave degradation might not markedly change the chemical components of the polysaccharides. High-molecular-weight polysaccharides from P. cruentum had no obvious antioxidant activity, but low-molecular-weight fragments after degradation exerted an inhibitory effect on oxidative damage. The 6.55-kDa fragment had stronger antioxidant activity than the 60.66 and 256-kDa fragments.     

Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ~97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ~45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.     

Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34-kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38-kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A-gold complexes demonstrated that the 38-kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34-kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34-kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.     

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.     

Purification, characterization and immunochemical properties of a novel 60-kDa protein of Vibrio anguillarum strains

Vibrio anguillarum strains expressed increased amounts of a novel 60-kDa protein when cells were grown at physiologically elevated temperatures. The relative amounts of the 60-kDa protein were unaltered by changes in osmolarity or ionic concentration of the growth medium in cells grown at optimal growth temperatures. The N-terminal amino acid sequence analysis of the V. anguillarum 60-kDa protein showed extensive (94–89%) sequence identity with the 60-kDa heat shock protein of Yersinia enterocolitica and with Serratia rubidaea GroEL protein. Monoclonal antibodies against the Y. enterocolitica chaperonin reacted with the 60-kDa protein from V. anguillarum strains, and with a temperature-induced protein of similar molecular mass in other Gram-negative pathogens of fish.