Glyceraldehyde-3-phosphate Dehydrogenase Protein Research Articles

RNA Binding of GAPDH Controls Transcript Stability and Protein Translation in Acute Myeloid Leukemia.

Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.

Oral administration of Lactobacillus delbrueckii subsp. lactis LDL557 attenuates airway inflammation and changes the gut microbiota in a Der p-sensitized mouse model of allergic asthma.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder affecting up to 20% of children in developed countries. Although probiotics have shown promise as adjuvant treatments for AD, their mechanisms are not well understood. Building upon our previous studies, we investigated whether Lactobacillus gasseri and its moonlighting glyceraldehyde 3-phosphate dehydrogenase (GAPDH), namely LGp40, could be beneficial in AD management. In AD mouse models (SKH and C57BL/6J mice) with ovalbumin (OVA) and Dermatophagoides pteronyssinus (Der p) allergens, aligning with the "outside-in" and "inside-out" hypotheses, we administered L. gasseri orally and LGp40 intraperitoneally to investigate their protective effects. The evaluation involved measuring physiological, pathological, and immune function parameters. To delve deeper into the detailed mechanism of LGp40 protection in AD, additional assays were conducted using human skin keratinocytes (HaCaT) and monocytes (THP1) cell lines. L. gasseri and LGp40 enhanced skin barrier function and increased skin moisture retention. They also led to reduced infiltration of Langerhans cells in the dermis and mitigated skewed Th2 and Th17 immune responses. Moreover, LGp40 inhibited allergen-induced keratinocyte apoptosis through the blockade of the caspase-3 cascade and reduced the NLR family pyrin domain containing 3 (NLRP3) inflammasome in macrophages. These inhibitions were achieved through the activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway. The results of this study provide a novel insight into the mechanism of action of probiotics in the prevention and treatment for allergic disorders through the moonlighting GAPDH protein.

NTRC regulates CP12 to activate Calvin–Benson cycle during cold acclimation

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

Exploration of Annona muricata (Annonaceae) in the Treatment of Hyperlipidemia through Network Pharmacology and Molecular Docking

Soursop (Annona muricata L.) is one of the plants that have antihyperlipidemic effects, but its underlying mechanism of action remains unknown. Previous investigations used TCMSP, KNApSAcK, ETCM, SwissTargetPrediction, SuperPred, CTD, and TTD to identify potential targets of soursop as antihyperlipidemic. Therefore, this study aims to explore soursop active compounds and demonstrate their mechanisms against hyperlipidemia through network pharmacology and molecular docking. OB and drug-likeness properties of the compounds from A. muricata were screened based on Lipinski’s Ro5 (Lipinski’s Rule of Five) parameters. Subsequently, the network of the compound–target–disease–pathways was constructed using Cytoscape. The target PPI (protein-protein interaction) network was built using STRING and the core targets were analyzed using GO with KEGG. The main active compounds against the targets were confirmed by molecular docking analysis. Based on the results, 158 compounds were identified in A. muricata, and the human body was found to absorb 56. It was discovered that 20 compounds were associated with cholesterol disease. The highest degree of the disease pathway of target compounds disease was annomuricin, XDH, myocardial ischemia, and metabolic pathways, respectively. The PPI showed GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein also has the highest degree. BP, CC, MF, and KEEG enrichments that play important roles are the response to drugs, plasma membranes, protein binding, and metabolic pathways. The molecular docking experiment confirmed the correlation between ligands and receptors (quercetin-XDH, coclaurine-ADRB3, fisetin, and robinetin-XDH) with binding energies of –9.3; –8.9; and –8.8 kcal mol–1, respectively. The interactions between ligands and receptors are hydrogen, alkyl, Pi-alkyl, Pi-sigma, and van der Waals bonds. It was discovered that A. muricata provided therapeutic effects, involving multi-compounds, multi-targets, multi-diseases, and multi-pathways as well as deep insight into the pathogenesis of hyperlipidemia. This can be used to design new drugs and develop novel therapies to treat hyperlipidemia.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin–Benson–Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde−3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

Effects of the Cobalt-60 gamma radiation on Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase

Purpose Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of the glycolytic pathway, can play a physiological regulatory role and vital other roles in metabolism. This study investigated the effects of gamma radiation generated by Cobalt-60 source on GAPDH activity and protein levels in Pichia pastoris as an eukaryotic organism model. Materials and methods After purification of the GAPDH from P. pastoris, in vitro effects of irradiation to the dose of 2 Gy, using Cobalt-60 at the dose rate of 0.25 Gy/min, on activity and kinetic parameters were investigated. In vivo effects of gamma exposition (dose of 5 Gy) on P. pastoris GAPDH and on reactive oxygen species (ROS) markers were also explored. Results and conclusions The in vitro irradiation of the purified GAPDH reduces the specific activity and the maximum velocity (V max) without alteration of substrates binding (K m). No changes occurred in the specific activity and in kinetic parameters when P. pastoris cells were exposed to Cobalt-60 source. However, this in vivo irradiation of cells produced a significant increase of the GAPDH protein level. The changes of GAPDH activity and the increase of the enzyme population as a target for gamma radiation exposure will play a role in cells adaptation under stress conditions. On the other hand, the increase of malondialdehyde and carbonyl contents and the enhancement of catalase and superoxide dismutase in irradiated cells have been noticed. The antioxidant system can play an important role in the protection of P. pastoris GAPDH against the gamma induced-ROS damage. This is the first report of the P. pastoris GAPDH as a physiological target of gamma exposition.

Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3′-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.

Capture and Identification of Dual Specificity Mitogen-Activated Protein Kinase Kinase 7 as a Direct Proteomic Target of Berberine to Affect the c-JunN-Terminal Kinase Pathway

Capture and Identification of Dual Specificity Mitogen-Activated Protein Kinase Kinase 7 as a Direct Proteomic Target of Berberine to Affect the c-JunN-Terminal Kinase Pathway

Glyceraldehyde‐3‐phosphate dehydrogenase protects smooth muscle cells against oxidative/genotoxic stress by activation of the apurinic/apyrimidinic endonuclease I pathway

The role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential regulator of cell death was initially interrogated by our laboratory as it pertains to the protection and stimulation of DNA repair that comes from the direct binding of nuclear GAPDH to apurinic/apyrimidinic endonuclease 1 (Ape1). To further elucidate this mechanism, we set to perturbate the GAPDH system in vascular smooth muscle cells (SMC) using a lentiviral GAPDH-overexpressing vector and cells were treated with etoposide (10-240uM), a pro-oxidant and genotoxic compound. Basal GAPDH and Ape1 protein levels were measured via Western Blotting. Etoposide (20 uM) decreased GAPDH and Ape1 protein levels at 54±7% and 42±7%, respectively. Etoposide increased phospho-histone H2A.X (S139) (double-strand DNA damage marker), and cleaved caspase-3 (Asp 175) (apoptosis marker) levels by 170±17% and 230±15%, respectively. Etoposide induced cell apoptosis in dose-dependent manner (Cell Death Elisa). Etoposide induced appearance of oxidized GAPDH in cell nuclear and cytosolic compartments (immunofluorescence with oxidized GAPDH antibody, 4A1 clone) and Etoposide also markedly suppressed GAPDH-Ape1 binding in the cell nucleus (proximity ligation assay). Cell transfection with GAPDH-overexpressing virus increased GAPDH protein, decreased Etoposide-induced cytotoxicity (live cell assay with Incucyte Sx5/Cytotox Red) and suppressed Etoposide-elicited caspase3/7 activation (Incucyte Caspase 3/7 Red dye). GAPDH overexpression reduced Etoposide-induced apoptosis and decreased phospho-histone H2A.X levels. In summary, we have created an efficient oxidative/genotoxic stress model in vascular SMC to analyze DNA damage and its mechanistic role in cell death and apoptosis-associated pathologies. We show that Etoposide induced a potent cytotoxic/genotoxic stress in SMC including DNA damage, and induction of cell apoptosis and these effects were associated with oxidation of GAPDH, reduction in GAPDH-Ape1 binding and marked Ape1 endonuclease downregulation. GAPDH overexpression prevented Etoposide-induced cytotoxicity and apoptosis potentially via stimulation of Ape1/DNA repair pathway.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

The possible effect of silver nanoparticles on glyceraldehyde‐3‐phosphate dehydrogenase activity and formation of amyloid‐like aggregates in MCF‐7 cell line

Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox-sensitive enzyme involved in apoptosis-related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF-7 cells were treated with different concentrations (0, 3, 22, and 200 μg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose-dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 μg/ml) and during short-term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 μg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose-dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)-related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.

Skin Delivery of siRNA Using Sponge Spicules in Combination with Cationic Flexible Liposomes

We report the topical administration of sponge Haliclona sp. Spicules (SHS) combined with cationic flexible liposomes (CFL) to increase the delivery of small interfering RNA (siRNA) into viable skin cells in vitro and in vivo. SHS can be applied topically as novel microneedles to overcome skin barrier by creating plenty of new microchannels in stratum corneum. Subsequently, well-designed CFL can be also utilized topically as nanocarriers to overcome skin cells membrane by delivering siRNA to skin deep layers through these microchannels and thereby facilitating their cell internalization. The topical application of SHS in combination with CFL (0.05% of lipids, w/v), referred to as CFL(0.05%), enhanced siRNA skin penetration in vitro by 72.95 ± 2.97-fold compared to control group (p < 0.001). Further, the topical application of SHS in combination with CFL(0.05%) on female BALB/c mice skin resulted in 29.21% ± 1.41% of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown at all application area in vivo, which was not significantly different from the GAPDH protein knockdown rate in the subcutaneous injection center. However, the high knockdown rate only appears in the vicinity (<0.5 cm) of the injection center. In sum, this study provides a promising strategy of topical delivery of siRNA by the combined used of SHS and well-designed CFL.

Atheroprotective Effects Induced by Glyceraldehyde‐3′‐phosphate Dehydrogenase (GAPDH) in Murine Model of Atherosclerosis

GAPDH was shown to protect smooth muscle cells (SMC) against oxidant‐induced apoptosis via stimulation of DNA repair. GAPDH levels were reduced in atherosclerotic plaque SMC and therefore we hypothesized that SMC‐specific GAPDH overexpression will reduce atherosclerosis. GAPDH protein levels were increased &gt;2‐fold (P&lt;0.05) in aortas isolated from Apoe‐null mice with SM22α promoter‐driven GAPDH overexpression (SM‐GAPDH mice) compared to Apoe‐null controls. SM‐GAPDH mice have BW and BP similar to control. Aortic circumference, lumen area and media area were not changed by GAPDH overexpression. SMC GAPDH did not change pulse wave velocity (SM‐GAPDH, 1.1±0.1 m/s, control, 1.2±0.3 m/s) (ultrasound imaging) as well as endothelium‐dependent and ‐independent vascular responses to PE, Ach or SNP (organ chamber assay with thoracic aortic rings). High‐cholesterol fed SM‐GAPDH had reduced atherosclerotic burden (H&amp;E‐stained aortic valve cross‐sections: 28±3% decrease, P&lt;0.05), elevated plaque SMC levels (IHC for a‐SM actin, 3.1‐fold increase, P&lt;0.01; IHC for calponin, 3.8‐fold increase, P&lt;0.05), increased plaque collagen (Trichrome, 2.1‐fold increase, P&lt;0.05), reduced plaque SMC apoptosis (TUNEL assay with IHC for calponin, 3.2‐fold decrease, IHC for cleaved caspase‐3, 2.6‐fold decrease both are P&lt;0.05) without changes in macrophages (IHC with Mac3, P=NS). Aortas isolated from SM‐GAPDH have increased expression of Ape1 endonuclease (major DNA repair enzyme, 31±5% increase, P&lt;0.05), reduced cleaved PARP (apoptotic marker, 74±12% decrease, P&lt;0.001) and decreased oxidative DNA damage (AP sites assay, 2.5‐fold decrease, P&lt;0.05; pSer139 histone H2AX, 33±3% decrease, P&lt;0.05) compared to controls. Plaques in SM‐GAPDH mice had thicker SMC‐rich fibrous plaque cap (3.5‐fold increase, P&lt;0.05) and decreased area of necrotic core (1.8‐fold reduction, P&lt;0.05) suggesting enhanced plaque stability. These data taken together with our in vitro findings indicate that Ape1 upregulation mediates GAPDH effect on DNA and apoptosis. Our results suggest that stimulation of GAPDH/Ape1 axis is a novel potential anti‐atherosclerotic therapy.Support or Funding InformationStudy was supported by NIH/NHLBI 1R01HL142796‐01A1 (SS), AHA Grant‐in‐aid (SS) and NIH/NHLBI 7R01HL070241 (PD).

Smooth Muscle Specific Glyceraldehyde‐3′‐phosphate dehydrogenase (GAPDH) Reduces DNA Damage, Decreases Cell Apoptosis, Suppresses Atherosclerosis and Promotes the Stable Plaque Phenotype

GAPDH is a glycolytic enzyme with a multiple glycolysis‐unrelated functions. We have shown that GAPDH protected smooth muscle cells (SMC) against oxidant‐induced apoptosis via upregulation of Ape1 endonuclease, the major DNA repair enzyme. We also found that GAPDH levels were reduced in atherosclerotic plaque SMC. We hypothesized that SMC‐specific GAPDH overexpression will reduce atherosclerosis. We generated Apoe‐null mice with SM22α promoter‐driven GAPDH overexpression (SM‐GAPDH mice). GAPDH protein levels were increased &gt;2‐fold (P&lt;0.05) in aortas isolated from SM‐GAPDH vs. Apoe‐null controls. SM‐GAPDH mice have BW and BP similar to control. Aortic circumference, lumen area and media area were not changed by GAPDH overexpression. SMC GAPDH did not change pulse wave velocity (SM‐GAPDH, 1.1±0.1 m/s, control, 1.2±0.3 m/s) (ultrasound imaging) as well as endothelium‐dependent and ‐independent vascular responses to PE, Ach or SNP (organ chamber assay with thoracic aortic rings). High‐cholesterol fed SM‐GAPDH had reduced atherosclerotic burden (H&amp;E‐stained aortic valve cross‐sections: 28±3% decrease, P&lt;0.05), elevated plaque SMC levels (IHC for a‐SM actin, 3.1‐fold increase, P&lt;0.01; IHC for calponin, 3.8‐fold increase, P&lt;0.05), increased plaque collagen (Trichrome, 2.1‐fold increase, P&lt;0.05), reduced plaque SMC apoptosis (TUNEL assay with IHC for calponin, 3.2‐fold decrease, IHC for cleaved caspase‐3, 2.6‐fold decrease both are P&lt;0.05) without changes in macrophages (IHC with Mac3, P=NS). Aortas isolated from SM‐GAPDH have increased Ape1 endonuclease (31±5% increase, P&lt;0.05), reduced cleaved PARP (apoptotic marker, 74±12% decrease, P&lt;0.001) and decreased oxidative DNA damage (AP sites assay, 2.5‐fold decrease, P&lt;0.05; pSer139 histone H2AX, 33±3% decrease, P&lt;0.05) compared to controls. Plaques in SM‐GAPDH had thicker SMC‐rich fibrous plaque cap (3.5‐fold increase, P&lt;0.05) and decreased area of necrotic core (1.8‐fold reduction, P&lt;0.05) suggesting enhanced plaque stability. These data taken together with our in vitro findings indicate that Ape1 upregulation mediates GAPDH effect on DNA and apoptosis. Stimulation of GAPDH/Ape1 axis is a novel potential anti‐atherosclerotic therapy.Support or Funding InformationNIH/NHLBI R01HL070241 (P.D.), American Heart Association Grant‐in‐Aid 13GRNT17230069 (S.S.), and University of Missouri School of Medicine Bridge Fund (S.S.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

Cloning and Expression Profile of Glyceraldehyde-3-Phosphate Dehydrogenase in Haemaphysalis flava (Acari: Ixodidae).

Haemaphysalis flava (Acari: Ixodidae) harbors pathogenic microorganisms and transfers these to hosts during blood feeding. Proteomic analysis in the midgut contents of H. flava detected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and contig 1683 was retrieved as a GAPDH gene fragment by searching our previous transcriptomic library. In the study, the 5' and 3' ends of contig 1683 were cloned by rapid amplification of cDNA ends (RACE) and a full length, 1340 bp cDNA of Hf-GAPDH was obtained. The open-reading frame had 999 bp and coded for 333 amino acids. Hf-GAPDH was predicted to have an N-terminal NAD binding domain and a C-terminal glyceraldehyde dehydrogenase catalytic domain. The molecular structure of Hf-GAPDH was analyzed and the evolutionary relationship also established. The GAPDH protein sequence was conserved among ticks. The expression pattern of Hf-GAPDH, analyzed by real-time PCR, significantly differed among life phases, feeding stages, and tissues. As the ticks grew, the expression level of Hf-GAPDH was up-regulated. The expression levels of Hf-GAPDH in salivary glands and midguts from half-engorged ticks were lower than the same tissues from engorged ticks. This study will provide reference data for the follow-up verification of the GAPDH-related function and the feasibility as a potential anti-tick vaccine.

Correlation study of GAPDH, Bcl-2, and Bax protein immunoexpression in patients with colorectal adenocarcinoma.

IntroductionColorectal cancer (CRC) is the third and second most commonly diagnosed cancer worldwide in males and females, respectively. Despite prominent progress in diagnosis and treatment, the recurrence rates are still high. A tumour hypoxic environment leads to an increase in glycolytic metabolism. The crucial intermediate component of glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), could play a significant role in cancer progression. An increased level of GAPDH has been described in oncogene-induced transformation and anti-apoptotic function. In other studies, GAPDH has been involved in apoptosis induction.AimWe examined colorectal adenocarcinoma samples to assess the immunoexpression of GAPDH protein. We also evaluated the correlation between the expression of GAPDH protein and apoptotic parameters including expression of Bcl2 and Bax.Material and methodsParaffin sections were incubated for 60 min with primary antibody against GAPDH, Bcl-2, and Bax.ResultsResults of our study have shown that GAPDH expression in colorectal cancer is upregulated. We revealed significant positive correlation between expression of this protein and grade and size of tumour, and regional lymph node involvement. In the case of apoptosis-associated proteins, e.g. Bcl-2 and Bax, we found negative correlations between expression of these proteins and grade and size of tumour, lymphovascular invasion, and regional lymph node involvement. Finally, we demonstrated that GAPDH up-regulation is connected with down-regulation in Bcl-2 and Bax.ConclusionsUp-regulation of GAPDH protein and down-regulation of Bcl-2 and Bax may result in increased of cancer.

An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction

An appropriate loading control is critical for Western blot analysis. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction. This study was undertaken to look for an appropriate loading control for western blot analysis of ischemic myocardium. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. The heart tissue samples from different areas and time points after surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. However, the protein level of GAPDH was stable in the Rhesus monkey model. We concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.

Abstract 5430: GAPDH loss in a tumorigenic human glioblastoma cell line

Abstract Background: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, multifunctional 37 kDa enzyme best known for catalyzing the sixth step in glycolysis. GAPDH expression is so consistent that it has been granted “housekeeping” status, and is used as a standard (along with β-actin) for normalizing Western blots. While increased GAPDH expression has been described in hypoxia, diabetes, and some cancers, decreased expression is rare. Here we describe a new human glioblastoma cell line that exhibits GAPDH loss, while maintaining a high degree of malignancy. Methods: Primary cultures of human glioblastoma cells were initiated and maintained in DMEM with 10% FBS. This particular cell line (designated E297) grew readily, and was passaged more than 40 times. E297 cells were confirmed to be negative for HIV, HTLV, hepatitis B and C, CMV, and mycoplasma contamination. Cells in exponential growth were stained for routine markers, and studied by DNA flow cytometry. GAPDH expression was studied by Western blot analysis using polyclonal rabbit anti-human GAPDH IgG (Abcam, Rosemont, IL), with HeLa, U138, U87 and U251 cells (as well as β-actin staining) as positive controls. For intracranial implantation into Wistar-Furth rats (male, 10-11 weeks), cells were suspended at 20 x 106 cells/ml. Using aseptic technique, rats were anesthetized, and placed into a stereotactic frame. A burr hole was drilled 3 mm right of the bregma and 25 µl of suspension injected (5 mm depth) over 10 min. by Hamilton syringe. Rats were studied by MRI and euthanized when symptomatic. Results: E297 cells have glial/epithelioid morphology, with a doubling time of 24 + 2 hours in logarithmic growth phase, with a single DNA subpopulation with 28% S phase. They stain positively for GFAP, vimentin, bFGF, c-myc and p53, but fail to express GAPDH. 10/10 animals implanted intracerebrally developed tumors, becoming symptomatic and requiring sacrifice at 25 days (mean). Loss of GAPDH expression was confirmed by Western blot analysis. Conclusions: The E297 human glioblastoma cell line is highly aggressive and proliferates rapidly. It is tumorigenic in nonimmunosuppressed rats but lacks GAPDH protein. We conclude that GAPDH expression is not essential for glioblastoma cell proliferation under routine culture conditions, and care needs to be taken when using GAPDH as a normalization standard for Western blots of cancer cells. Citation Format: Zachary Gaertner, Yi Lu, Jinsuh Kim, Gayatry Mohapatra, Ankit Mehta, Sanjani Lakka, Herbert Engelhard. GAPDH loss in a tumorigenic human glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2017-5430

Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency

Mice with the hypomorphic AIF-Harlequin mutation exhibit a highly heterogeneous mitochondriopathy that mostly affects respiratory chain complex I, causing a cerebral pathology that resembles that found in patients with AIF loss-of-function mutations. Here we describe that the antidiabetic drug pioglitazone (PIO) can improve the phenotype of a mouse Harlequin (Hq) subgroup, presumably due to an inhibition of glycolysis that causes an increase in blood glucose levels. This glycolysis-inhibitory PIO effect was observed in cultured astrocytes from Hq mice, as well as in human skin fibroblasts from patients with AIF mutation. Glycolysis inhibition by PIO resulted from direct competitive inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, GAPDH protein levels were reduced in the cerebellum and in the muscle from Hq mice that exhibited an improved phenotype upon PIO treatment. Altogether, our results suggest that excessive glycolysis participates to the pathogenesis of mitochondriopathies and that pharmacological inhibition of glycolysis may have beneficial effects in this condition.