Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Expression of a heptelidic acid-insensitive recombinant GAPDH from Trichoderma virens, and its biochemical and biophysical characterization.

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the "vir" cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec-1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the "vir" cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the "vir" cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

Complementary Oligonucleotide Conjugated Multicolor Carbon Dots for Intracellular Recognition of Biological Events.

By using complementary DNA sequences as surface ligands, we selectively allow two individual diffusing "dual-color" carbon dots to interact in situ and in vitro. Spontaneous nanoscale oxidation of surface-abundant nitroso-/nitro-functionalities leads to two distinctly colored carbon dots (CD) which are isolated by polarity driven chromatographic separation. Green- and red-emitting carbon dots (gCD and rCD) were decorated by complementary single-stranded DNAs which produce a marked increase in the fluorescence emission of the respective carbon dots. Mutual colloidal interactions are achieved through hybridization of complementary DNA base pairs attached to the respective particles, resulting in quenching of their photoluminescence. The observed post-hybridization quenching is presumably due to a combined effect from an aggregation of CDs post duplex DNA formation and close proximity of multicolored CDs, having overlapped spectral regions leading to a nonradiative energy transfer process possibly released as heat. This strategy may contribute to the rational design of mutually interacting carbon dots for a better control over the resulting assembly structure for studying different biological phenomenon including molecular cytogenetics. One of the newly synthesized CDs was successfully used to image intracellular location of GAPDH mRNA using an event of change in fluorescence intensity (FI) of CDs. This selectivity was introduced by conjugating an oligonucleotide harboring complementary sequence to GAPDH mRNA. FI of this conjugated carbon dot, rCD-GAPDH, was also found to decrease in the presence of Ca2+, varied in relation to H+ concentrations, and could serve as a tool to quantify the intracellular concentrations of Ca2+ and pH value (H+) which can give important information about cell survival. Therefore, CD-oligonucleotide conjugates could serve as efficient probes for cellular events and interventions.

Quantification of cDNA on GMR biosensor array towards point-of-care gene expression analysis.

Gene expression analysis at the point-of-care is important for rapid disease diagnosis, but traditional techniques are limited by multiplexing capabilities, bulky equipment, and cost. We present a gene expression analysis platform using a giant magnetoresistive (GMR) biosensor array, which allows multiplexed transcript detection and quantification through cost-effective magnetic detection. In this work, we have characterized the sensitivity, dynamic range, and quantification accuracy of Polymerase chain reaction (PCR)-amplified complementary DNA (cDNA) on the GMR for the reference gene GAPDH. A synthetic GAPDH single-stranded DNA (ssDNA) standard was used to calibrate the detection, and ssDNA dilutions were qPCR-amplified to obtain a standard curve. We demonstrate that the GMR platform provides a dynamic range of 4 orders of magnitude and a limit of detection of 1 pM and 0.1 pM respectively for 15 and 18-cycle amplified synthetic GAPDH PCR products. The quantitative results of GMR analysis of cell-line RNA were confirmed by qPCR.

Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study.

Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin ß-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin ß-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MSE, was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture.

Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis. It is also referred to as a moonlighting protein as it has many diverse functions like regulation of apoptosis, iron homeostasis, cell-matrix interactions, adherence to human colon etc. apart from its principal role in glycolysis. Lactobacilli are lactic acid bacteria which colonize the human gut and confer various health benefits to humans. In the present study, we have cloned, expressed and purified the GAPDH from Lactobacillus acidophilus to get a recombinant product (r-LaGAPDH) and characterized it. Size exclusion chromatography shows that r-LaGAPDH exists as a tetramer in solution and have a mucin binding and hemagglutination activity indicating carbohydrate like binding adhesion mechanism. Fluorescence spectroscopy studies showed an interaction of r-LaGAPDH with mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine with a Kd of 3.6±0.7×10(-3)M, 4.34±0.09×10(-3)M, 4±0.87×10(-3)M and 3.7±0.28×10(-3)M respectively. We hope that this preliminary data will generate more interest in further elucidation of the roles of GAPDH in the adhesion processes of the bacteria.

Millisecond duration pulses for flow-through electro-induced protein extraction from E. coli and associated eradication.

Pulsed electric fields are used to induce membrane permeabilization on cells. In the case of species with cell wall (yeasts, microalgae), it was previously shown that when the pulse duration was several ms long, this resulted in a cytoplasmic soluble protein slow leakage. In this work, we show that a similar consequence can be obtained with different strains of E. coli. Experimental evidences of a resulting wall alteration are described. Pre-industrial flow process pilots are used. As the membrane electropermeabilization can be irreversible by applying a proper choice of the pulse parameters, this approach is used for bacterial inactivation in flow process. It is observed that sub-millisecond pulse trains are more cost effective than longer ones.

Nuclear accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase in cadmium-stressed Arabidopsis roots.

NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in the glycolytic pathway. It has been widely demonstrated that mammalian GAPDH, in addition to its role in glycolysis, fulfills alternative functions mainly linked to its susceptibility to oxidative posttranslational modifications. Here, we investigated the responses of Arabidopsis (Arabidopsis thaliana) cytosolic GAPDH isoenzymes GAPC1 and GAPC2 to cadmium-induced stress in seedlings roots. GAPC1 was more responsive to cadmium than GAPC2 at the transcriptional level. In vivo, cadmium treatments induced different concomitant effects, including (1) nitric oxide accumulation, (2) cytosolic oxidation (e.g. oxidation of the redox-sensitive Green fluorescent protein2 probe), (3) activation of the GAPC1 promoter, (4) GAPC1 protein accumulation in enzymatically inactive form, and (5) strong relocalization of GAPC1 to the nucleus. All these effects were detected in the same zone of the root tip. In vitro, GAPC1 was inactivated by either nitric oxide donors or hydrogen peroxide, but no inhibition was directly provided by cadmium. Interestingly, nuclear relocalization of GAPC1 under cadmium-induced oxidative stress was stimulated, rather than inhibited, by mutating into serine the catalytic cysteine of GAPC1 (C155S), excluding an essential role of GAPC1 nitrosylation in the mechanism of nuclear relocalization, as found in mammalian cells. Although the function of GAPC1 in the nucleus is unknown, our results suggest that glycolytic GAPC1, through its high sensitivity to the cellular redox state, may play a role in oxidative stress signaling or protection in plants.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 µM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly.

Novel inhibitors of glyceraldehyde-3-phosphate dehydrogenase: covalent modification of NAD-binding site by aromatic thiols.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) is a glycolytic enzyme catalyzing the formation of 1,3-diphosphoglycerate from glyceraldehyde-3-phosphate and inorganic phosphate. In cooperation with E3 ubiquitin-kinase Siah1, GAPDH directly participates in the apoptotic death of neurons in Parkinson's disease. Potential GAPDH inhibitors were screened in silico, and three compounds with high affinity to the NAD-binding site and theoretically capable of forming a disulfide bond with amino acid residue Cys149 were found among cysteine and glutathione derivatives. The inhibitory effect of these compounds was tested on GAPDH from rabbit muscles using isothermal calorimetry and kinetic methods. As a result of experimental screening, we selected two compounds that inhibit GAPDH by forming disulfide bonds with the Cys149 residue in the enzyme active site. Since Cys149 is the key residue not only for the catalyzed reaction, but also for interaction with Siah1, the compounds can be assumed to inhibit the formation of the proapoptotic complex GAPDH-Siah1 and therefore have potential effect against Parkinson's disease.

DNA-enrichment microfluidic chip for chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency.

Investigation of glyceraldehyde-3-phosphate dehydrogenase from human sperms.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus.

The NAD+-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants (K(m)) for NAD+ and D-glyceraldehyde-3-phosphate were 92.0 microM and 73.4 microM, respectively. The maximal velocity (V(max)) of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 degrees C. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform (pI 7.9).

Proteomics analysis of the tombusvirus replicase: Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication.

Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown approximately 35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of human liver glyceraldehyde-3-phosphate dehydrogenase.

Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region.

P but not R-axis interface is involved in cooperative binding of NAD on tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

Homotetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three non-equivalent P, R, and Q interfaces. In our previous study, negative cooperativity in NAD binding to wild-type GAPDH was interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding sites in each dimer. Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD binding. Based on the molecular modeling of the substitutions and the fact that the most extensive inter-subunit interactions are formed across the P-axis interface of the tetramer, it was postulated that both dimeric mutant GAPDHs were of O-P type. Therefore, the P-axis interface was assumed to play a major role in causing cooperativity in NAD binding.Here, two other mutant GAPDHs, Y46G/R52G and D282G, have been studied. Using small angle X-ray scattering, the dimeric form of the D282G mutant GAPDH is shown to be of O-R type whereas both dimeric mutant GAPDHs Y46G/R52G and Y46G/S48G are of O-P type. Similarly to dimeric Y46G/S48G mutant GAPDH, the dimeric Y46G/R52G mutant GAPDH exhibits positive cooperativity in NAD binding. On the other hand, no significant cooperativity in NAD binding to the dimeric form of the D282G mutant GAPDH is observed, whereas its tetrameric counterpart exhibits negative cooperativity, similarly to the wild-type enzyme. Altogether, the results support the view that the P-axis interface is essential in causing cooperativity in NAD binding by transmitting the structural information induced upon cofactor binding from one subunit to the other one within O-P/Q-R dimers in contrast to the R-axis interface, which does not transmit structural information within O-R/Q-P dimers. The absence of activity of O-P and O-R dimer GAPDHs is the consequence of a pertubation of the conformation of the active site, at least of the nicotinamide subsite, as evidenced by the absence of an ion pair between catalytic residues C149 and H176 and the greater accessibility of C149 to a thiol kinetic probe.