Human Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Discovery of a spirocyclic 3-bromo-4,5-dihydroisoxazole covalent inhibitor of hGAPDH with antiproliferative activity against pancreatic cancer cells

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

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.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

Simple SummaryCancer cells largely use glycolysis to obtain both chemical energy (ATP) and metabolic intermediates for anabolic reactions, and several studies proved that the blockage of the glycolytic pathway is an efficient anticancer strategy. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key tetrameric glycolytic enzyme, has raised considerable attention in recent years as a potential drug target in pathological conditions in which glycolytic flux has a crucial role. In cancers, a recognized involvement of the Warburg effect, as a key mechanism for cancer-cell energetic metabolism, favouring tumour progression and invasion, has contributed to highlight human GAPDH (hGAPDH) as an effective drug target to specifically hit cancer cells exhibiting metabolic dependence on glycolysis, without significantly affecting normal cells. In this study, we tested the effects of 3-bromo-isoxazoline derivatives specifically designed to bind and inhibit GAPDH activity, in pancreatic ductal-adenocarcinoma cells.A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Detection and quantification of human-specific mRNA from hepatocyte-like cells derived from dental pulp using real-time polymerase chain reaction

ObjectivesWe quantified viable hepatocyte-like cells (HLCs) administered via portal or tail veins in the livers and lungs of immunodeficient rats using real-time reverse transcription polymerase chain reaction (RT-PCR) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. MethodsImmunodeficient rats were infused with HLCs via portal or tail veins. mRNA was quantified based on the route of cell administration and the presence of liver injury. ResultsHuman-specific GAPDH mRNA primers detected 0.1 pg human RNA in 100 ng (1:106) of rat liver RNA. When infused into the portal vein, the quantity of HLC mRNA reduced to 5% 3 h after infusion. Most HLCs were entrapped in the lungs when infused via the tail vein and decreased to approximately 10% 6 h after infusion. A small number of HLCs made it to the liver but disappeared rapidly, regardless of liver injury. 24 h after infusion, viable HLCs were detected only in the lungs of rats with liver injury (P < 0.05). ConclusionsThe quantity of viable human cells in immunodeficient rats was estimated using real-time RT-PCR and primers specific to human mRNA.

Suitable primers for GAPDH reference gene amplification in quantitative RT-PCR analysis of human gene expression

In molecular biology, real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) has been commonly used for the evaluation of gene expression in human cells and tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is commonly used as a reference gene for the analysis of RT-qPCR experiments. However, recent studies have recognized several variants of GAPDH gene transcripts in human tissues. Thus, the application of the GAPDH gene in RT-qPCR amplification was also established with false interpretations of differential gene expression analysis. Thereof, a consistent amplification of reference gene transcript is essentially required from various tissues or cells treated under differential conditions. In this study, we designed a set of oligo primers for consistent amplification of the human GAPDH transcripts as a reference for human gene amplification. The designed primers for the GAPDH gene showed equalized amplification of GAPDH transcripts from human fibroblasts cells for differential gene expression analysis under various experimental conditions.

Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Å resolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c=341.007 Å, α = β = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.

Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

Structural Study of Monomethyl Fumarate-Bound Human GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al. published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at 2.29 Å. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between NAD+-bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the NAD+ co-substrate due to steric hindrance of the nicotinamide portion of the NAD+ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

Quadruplex real-time PCR for rapid detection of human alphaherpesviruses

Infections with the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) as well as with the varicella-zoster virus (VZV) may take a serious course. Thus, rapid and reliable detection of these alphaherpesviruses is urgently needed. For this, we established a qualitative quadruplex real-time polymerase chain reaction (PCR) covering HSV-1, HSV-2, VZV and endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR was validated with quality assessment samples and pre-characterized clinical samples including swabs, blood and cerebrospinal as well as respiratory fluids. For comparison, nucleic acids (NA) of selected samples were extracted manually and automatically. The protocol takes approx. 90min, starting with the preparation of NA until the report of results. The oligonucleotide and hydrolysis probe sequences specifically detect and distinguish HSV-1 (530nm), HSV-2 (705nm) and VZV (560nm) DNA. The detection limit was estimated with 100-500 copies/ml HSV-1 and HSV-2/VZV, respectively. All quality assessment samples as well as all the patient samples were classified correctly. Parallel detection of GAPDH (670nm) DNA was implemented to demonstrate correct sampling, but was uncertain in case of swabs. To this end, alphaherpesvirus-free human DNA was also added directly into the mastermix to exclude PCR inhibition. The established protocol for parallel detection and differentiation of alphaherpesviruses is fast, highly specific as well as rather sensitive. It will facilitate HSV-1/2 and VZV diagnostics and may be further improved by opening the 670nm channel for a combined extraction and PCR inhibition control.

GAPDH with NAD+-binding site mutation competitively inhibits the wild-type and affects glucose metabolism in cancer

BackgroundRapid utilization of glucose is a metabolic signature of majority of cancers, hence enzymes of the glycolytic pathway remain attractive therapeutic targets. Recent reports have shown that targeting the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an abundant, ubiquitous multifunctional protein frequently upregulated in cancer, affects cancer progression. Here, we report that a catalytically-deficient mutant-GAPDH competitively inhibits the wild-type, and disrupts glucose metabolism in cancer cells. MethodsUsing site-directed mutagenesis, the human GAPDH clone was mutated at one of the NAD+-binding sites, (i.e.) arginine (R13) and isoleucine (I14) to glutamine (Q13) and phenylalanine (F14), respectively. The inhibitory role of the mutant-GAPDH, and its effect on energy metabolism and cancer phenotype was determined using in vitro and in vivo models of cancer. ResultsThe enzymatically-dysfunctional mutant-GAPDH competitively inhibited the wild-type GAPDH in a cell-free system. In cancer cells, ectopic expression of the mutant-GAPDH, but not the wild-type, inhibited the glycolytic capacity of cellular-GAPDH, and led to the induction of metabolic stress accompanied by a sharp decline in glucose-uptake. Furthermore, expression of mutant-GAPDH affected cancer growth in vitro and in vivo. Mechanistically, structural analysis by bioinformatics revealed that the mutations at the NAD+-binding site altered the solvent-accessibility that perhaps affected the functionality of mutant-GAPDH. ConclusionMutant-GAPDH affects the enzymatic function of cellular-GAPDH and disrupts energy metabolism. General significanceOur findings demonstrate that a minimal mutation at the NAD+-binding site is sufficient to generate a competitive but dysfunctional GAPDH, and its ectopic expression inhibits the wild-type to disrupt glycolysis.

Moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is required for efficient hepatitis C virus and dengue virus infections in human Huh-7.5.1 cells

Hijacking of cellular biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle. In this study, the accumulating evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the host secretory pathway led us to hypothesize that this moonlighting enzyme could play a key role in the lifecycle steps of two important Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV). We used short interfering RNA (siRNA)-mediated knockdown of human GAPDH in Huh-7.5.1 cells- both pre- and post-HCV infection- to demonstrate that GAPDH is a host factor for HCV infection. siRNA-induced GAPDH knockdown performed pre-HCV infection inhibits HCV core production in infected cells and leads to a decrease in infectivity of the HCV-infected cell supernatants. siRNA-induced GAPDH knockdown performed post-HCV infection does not have an effect on HCV core abundance in infected cells, but does lead to a decrease in infectivity of the HCV-infected cell supernatants. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the infectivity of HCV-infected cell supernatants, suggesting a role for GAPDH during HCV post-replication steps. Interestingly, siRNA-induced GAPDH knockdown in HCV replicon-harbouring cells had no effect on viral RNA replication. Importantly, we confirmed the important role of GAPDH in the HCV lifecycle using Huh-7-derived stable GAPDH-knockdown clones. Finally, siRNA-induced GAPDH knockdown inhibits intracellular DENV-2 E glycoprotein production in infected cells. Collectively, our findings suggest that the moonlighting enzyme, GAPDH, is an important host factor for HCV infection, and they support its potential role in the DENV lifecycle.

Molecular basis for covalent inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by a 2‐phenoxy‐1,4‐naphthoquinone small molecule

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

Human GAPDH Is a Target of Aspirin's Primary Metabolite Salicylic Acid and Its Derivatives.

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death.

Overexpression of glyceraldehyde 3-phosphate dehydrogenase prevents neurovascular degeneration after retinal injury.

Ischemia and reperfusion (I/R) injury is a common cause of many vascular and neuronal diseases. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been found down-regulated or dysfunctional in several tissues upon I/R injury. To investigate the role of GAPDH in retinal I/R injury-induced neurovascular degeneration, the injured retinas of GAPDH transgenic (Tg) mice and wild-type (WT) littermates were analyzed. I/R injury induced neurovascular degeneration, energy failure, DNA damage, and necroptosis in the retinas of WT mice. In contrast, the GAPDH Tg mice showed resistance to all of these injury-induced abnormalities. In addition, I/R-induced effects were further examined in a neuroblastoma cell line and an endothelial cell line, which were transfected with a vector encoding human GAPDH or a control vector. After I/R challenge, energy failure, DNA damage, and elevation of receptor-interacting serine/threonine-protein kinase (RIP) 1/3 were observed in the cells transfected with the control vector. However, overexpression of GAPDH in these cells prevented the injury-induced RIP3 up-regulation by restoring energy production and preventing DNA damage. Together, the protective role of GAPDH in retinal neurovascular degeneration after I/R injury provides a better understanding of the underlying mechanism of I/R injury and a potential therapeutic target to attenuate I/R injury-related diseases.

Changes in the levels of Cryspovirus during in vitro development of Cryptosporidium parvum.

The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of Cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan. Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C. parvum sporozoites, followed by harvest of cells and culture medium at 2-, 24-, 48-, and 72-h post-infection. Changes in viral RNA levels were detected by RT-PCR using primers specific for RNA encoding the 40-kDa capsid protein (CP) or RNA-dependent RNA polymerase (RdRp). Parasite or host DNA was quantified by PCR specific for C. parvum or human glyceraldehyde-3-phosphate dehydrogenase (HuGAPDH). An internal standard (competitor) was incorporated into all assays as a control for PCR inhibition. Intracellular levels of C. parvum DNA increased between 2- and 48-h post-infection, and then decreased at 72 h. Culture medium overlying these C. parvum-infected cells displayed a similar increase in CP and RdRp signal, reaching peak levels at 48 h. However, the CP and RdRp levels in cellular RNA displayed only a modest increase between 2 and 48 h, and exhibited no change (CP) or decreased (RdRp) at 72 h. These data suggest that during the first 48 h of C. parvum in vitro development, Cryspovirus is released into the media overlying cells but remains at fairly constant levels within infected cells.

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.

The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) is regulated by myeloid zinc finger 1 (MZF-1) and is induced by calcitriol

Recently, new tissue-specific functions for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been discovered, aside from its archetypal function in glycolysis. This casts doubt on the legitimacy of using GAPDH as a normalization control for gene expression analysis. We report the binding of the myeloid zinc finger-1 (MZF-1) transcription factor to the human GAPDH promoter. Furthermore, we show that up-regulation of MZF-1 by 1,25-dihydroxyvitamin D3 (calcitriol) induces GAPDH in HS-5 stromal fibroblasts, while knockdown of MZF1 by shRNA leads to a concomitant reduction in GAPDH expression. This argues that MZF-1 regulates GAPDH, indicating a role for GAPDH in calcitriol-mediated signaling.

Determining the Effect of Dithiolethione Compounds on the Activity of Human Glyceraldehyde-3-Phosphate Dehydrogenase

Dithiolethione compounds, like anethole dithiolethione (ADT) have chemopreventive, cytoprotective, and antimitogenic effects. The chemopreventive effects are thought to be mediated by the activation of antioxidant response elements and of the tumor repressor PP2a. We have shown that ADT interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cancer cells usually exhibit increased glycolysis (Warburg effect) that generates ATP to meet their energy needs. Mounting evidence suggests that inhibition of glycolysis in cancer cells severely depletes ATP and may be an effective strategy in chemoprevention. We hypothesize that the chemopreventive effects of ADT may be in part due to inhibition of glycolysis via inhibition of GAPDH. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol-modifying agents. Therefore, we set out to examine how ADT affects GAPDH activity. Human GAPDH was overexpressed in bacterial cells and purified by ion-exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT and showed a concentration-dependent inhibition of GAPDH catalytic activity by ADT. In order to determine the exact mechanism of ADT modification of GAPDH we are coupling site-directed mutagenesis with enzymatic assay, and mass-spectrometry.This investigation was sponsored in part by NIH/NIGMS MARC U∗STAR T34 08663 National Research Service Award to UMBC and also by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program (JC) and UMBC DRIF SRAIS (EG).

A Peptide Derived from the Highly Conserved Protein GAPDH Is Involved in Tissue Protection by Different Antifungal Strategies and Epithelial Immunomodulation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis but also in non-metabolic processes, including transcription activation and apoptosis. We report the isolation of an hGAPDH (2-32) fragment peptide from human placental tissue exhibiting antimicrobial activity. The peptide was internalized by cells of the pathogenic yeast Candida albicans and initiated a rapid apoptotic mechanism, leading to killing of the fungus. Killing was dose-dependent, with 10 µg/ml (3.1 µM) and 100 µg/ml hGAPDH (2-32) depolarizing 45% and 90% of the fungal cells in a population, respectively. Experimental C. albicans infection induced epithelial hGAPDH (2-32) expression. Addition of the peptide significantly reduced the tissue damage as compared to untreated experimental infection. Secreted aspartic proteinases (Saps) activity of C. albicans was inhibited by the fragment at higher concentrations with an ED50 of 160 mg/l (50 μM) for Sap1p and 200 mg/l (63 μM) for Sap2p while Sap3 was not inhibited at all. Interestingly, hGAPDH (2-32) induced significant epithelial IL-8 and GM-CSF secretion and stimulated TLR4 expression at low concentrations independently of the presence of C. albicans without any toxic mucosal effects.In the future, the combination of different antifungal strategies, e.g. a conventional fungicidal with immunomodulatory effects and the inhibition of fungal virulence factors might be a promising treatment option.

Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.