Levels Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Availability of individual proteins for quantitative analysis in postmortem brains preserved in two different brain banks.

Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.

Role of sleep in asthenospermia induced by di (2-ethyl-hexyl) phthalate.

Di (2-ethyl-hexyl) phthalate (DEHP) mainly enters the human body through the digestive tract, respiratory tract, and skin. At the same time, it has reproductive and developmental toxicity, neurotoxicity, and so on, which can cause the decrease of sperm motility. Asthenospermia is also known as low sperm motility, and the semen quality of men in some areas of China is declining year by year. Interestingly, previous studies have shown that sleep disorders can also lead to asthenospermia. However, the relationship between sleep, DEHP, and asthenospermia is still unclear. Analysis of the National Health and Nutrition Examination Survey (NHANES) population database showed that DEHP was associated with sleep disorders, and subsequent experiments in mice and Drosophila indicated that DEHP exposure had certain effects on sleep and asthenospermia. Furthermore, we analyzed the Comparative Toxicogenomics Database (CTD) to find out the common signaling pathway among the three: hypoxia-inducible factor 1(HIF-1). Then Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to screen out the proteins that DEHP affected the HIF-1 pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), serine/threonine-protein kinase (AKT1), epidermal growth factor receptor (EGFR), and finally Western blot analysis was used to detect the expression levels of the three proteins. Compared with the control group, DEHP decreased the protein expression levels of GAPDH and AKT1 in the HIF-1 pathway, and caused sleep disorders and decreased sperm motility. This study provides preliminary evidence for exploring the mechanism among DEHP, sleep disorders, and asthenospermia.

Multi-omics analysis reveals GAPDH posttranscriptional regulation of IFN-γ and PHGDH as a metabolic checkpoint of microglia polarization

A “switch” in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-β triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.

Biliverdin Reductase B Protects Against Primaquine Induced Hemolysis - Identification of a Novel Anti-Oxidant Pathway in Murine Erythrocytes

Background: Biliverdin Reductase B (BlvrB) is the fourth most abundant enzyme in red blood cells (RBCs) and is widely expressed in other tissues. In normal heme catabolism, porphyrin is converted to biliverdin-alpha (by hemoxygenase (HO)) and then reduced to bilirubin-alpha (by biliverdin reductase A (BlvrA)). Bilirubin-alpha is then glucuronidated and excreted. The large body of literature investigating heme metabolism, bilirubin-based pathologies, and the oxidative biology of bilirubin/biliverdin in adults focuses almost exclusively on BlvrA and the alpha isomers of biliverdin and bilirubin. However, porphyrin is a tetrapyrrole with 4 distinct meso-carbon bridges, each of which can be broken to generate a distinct biliverdin isomer (alpha, beta, gamma, or delta). Although HO is exquisitely specific for the alpha-bond (generating only biliverdin-alpha), chemical coupled oxidation of heme yields all four biliverdin isomers. BlvrB (and not BlvrA) can reduce each of non-alpha biliverdin isomers into their corresponding bilirubin isomers (bilirubin-beta is prevalent in neonatal jaundice and kernicterus). BlvrB also reduces flavins and additional quinone metabolites. Despite BlvrB's abundance and evolutionary conservation, its only known function is a subtle effect on hematopoietic lineage fate (megakaryocyte vs. erythroid). BlvrB has no known function in mature RBCs. We hypothesized that BlvrB plays an important antioxidant role that would only become obvious when other redundant anti-oxidant pathways were limited. Methods: A novel BlvrB knockout mouse was generated on a C57BL/6 background. A model of primaquine (PQ) induced oxidative hemolytic anemia in G6PD deficient mice was used as a clinically relevant scenario in which compromised antioxidant biology leads to treatment limiting pharmacotoxicity in RBCs. BlvrB-KO mice were crossed with two strains of mice that had murine G6PD replaced with either the A- deficient variant of human G6PD (hG6PD(A-)) or the non-deficient form of human G6PD (hG6PD(ND)). Four mouse strains (i.e., hG6PD(ND) and hG6PD(A-) mice with or without BlvrB deletion) received either a 4-day course of PQ or control PBS injections. Anemia was monitored by hematocrit (HCT). Reticulocyte count (RET) was monitored by flow cytometry as CD71+, thiazole orange+ erythrocytes. Peripheral blood was subjected to high resolution metabolomics to elucidate biochemical changes. Results: All mouse strains were healthy at baseline, with normal measured hematological parameters (i.e., Hct 40, RET 1.5%). No change in HCT or RET was observed in hG6PD(ND) or BlvrB-KOxhG6PD(ND) mice receiving PQ or in any strain receiving PBS. PQ induced hemolytic anemia in hG6PD(A-) mice, with an average HCT of 32.7 (18% drop) with RET of 3.1%. A significant exacerbation of PQ induced hemolysis was observed in BlvrB-KOxhG6PD(A-) mice that had an average HCT of 22.5 (44% drop) with RET of 5.3%. (n= 11 mice per group, p <0.001 between groups). Blood from PQ-treated hG6PD(A-)xBlvrBKO mice showed defective glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase, increased levels of multiple long-chain acylcarnitines, altered redox metabolism, and specific increases of hypoxanthine and sphingosine-1-phosphate (S1P). Three separate experiments were performed with similar results. Discussion: These data demonstrate that BlvrB deletion exacerbates oxidative injury and suggest that BlvrB provides a novel and hitherto undescribed antioxidant pathway in RBCs. Because approximately half a billion humans exhibit some form of G6PD deficiency, these data are also directly relevant to a large population of humans, which were modeled using hG6PD(A-) mice. These studies also define the metabolic lesion in peripheral blood. For example, GAPDH is well known to be highly sensitive to inactivation by oxidative damage, resulting in impaired glycolysis. Changes in hypoxanthine and S1P are known to be associated with decreased RBC function and survival. Finally, altered carnitine metabolism is associated with repair of lipid peroxidation in RBCs via the Lands Cycle. Although the mechanism by which BlvrB facilitates antioxidation is not yet clear, we hypothesize it involves redox cycling of non-alpha isomers of biliverdin and bilirubin. However, the mechanisms may be through reduction of flavins and/or other endogenous substrates. Ongoing studies are testing these hypotheses.

Research on the oncogenic role of the house-keeping gene GAPDH in human tumors.

As an internal reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis. While increasing evidence suggests that GAPDH plays a crucial role in tumorigenesis of some cancers, no systematic analysis of GAPDH has been conducted. Here, we sought to analyze the expression of GAPDH and its oncogenic processes in pan-cancer. GAPDH was investigated in The Cancer Genome Atlas (TCGA) tumor types using several bioinformatic tools including Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), University of ALabama at Birmingham CANcer (UALCAN), cBio Cancer Genomics Portal (cBioPortal), and Search Tool for Recurring Instances of Neighbouring Genes (STRING) for the expression and relationships with prognosis and immune infiltration separately. Through our analysis, we measured the higher expression of GAPDH across the majority of TCGA tumors. GAPDH overexpression predicts poor survival in patients with tumors expressing a high level of GAPDH. Moreover, the genetic changes in GAPDH contributed to an increased mRNA expression. Additionally, GAPDH expression was negatively correlated with immune infiltration involving cancer-associated fibroblasts, neutrophil cell and endothelial. The house-keeping gene GAPDH might be a promising biomarker for pan-cancer prognosis. And GAPDH is not suitable as an internal reference gene for most cancer research, whether RNA or protein analyses.

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel, Anguilla anguilla.

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.

The intersection of metabolism and inflammation is governed by the intracellular topology of hexokinases and the metabolic fate of glucose.

Hexokinases (HKs) catalyze the first and irreversible step of glucose metabolism. Its product, glucose-6-phosphate (G-6P) serves as a precursor for catabolic processes like glycolysis for adenosine 5'-triphosphate (ATP) production and anabolic pathways including the pentose phosphate pathway (PPP) for the generation of intermediaries like nicotinamide adenine dinucleotide phosphate (NADPH) and ribulose-5-P. Thus, the cellular fate of glucose is important not only for growth and maintenance, but also to determine different cellular activities. Studies in immune cells have demonstrated an intimate linkage between metabolic pathways and inflammation, however the precise molecular mechanisms that determine the cellular fate of glucose during inflammation or aging are not completely understood. Here we discuss a study by De Jesus et al that describes the role of HK1 cytosolic localization as a critical regulator of glucose flux by shunting glucose into the PPP at the expense of glycolysis, exacerbating the inflammatory response of macrophages. The authors convincingly demonstrate a novel mechanism that is independent of its mitochondrial functions, but involve the association to a protein complex that inhibits glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase. We expand the discussion by comparing previous studies related to the HK2 isoform and how cells have evolved to regulate the mitochondrial association of these two isoforms by non-redundant mechanism.

AMP-activated protein kinase-dependent nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in senescent human diploid fibroblasts.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that participates in various cellular events, such as DNA repair and apoptosis. The functional diversity of GAPDH depends on its intracellular localization. Because AMP-activated protein kinase (AMPK) regulates the nuclear translocation of GAPDH in young cells and AMPK activity significantly increases during aging, we investigated whether altered AMPK activity is involved in the nuclear localization of GAPDH in senescent cells. Age-dependent nuclear translocation of GAPDH was confirmed by confocal laser scanning microscopy in human diploid fibroblasts (HDFs) and by immunohistochemical analysis in aged rat skin cells. Senescence-induced nuclear localization was reversed by lysophosphatidic acid but not by platelet-derived growth factor. The extracellular matrix from young cells also induced the nuclear export of GAPDH in senescent HDFs. An activator of AMPK, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), increased the level of nuclear GAPDH, whereas an inhibitor of AMPK, Compound C, decreased the level of nuclear GAPDH in senescent HDFs. Transfection with AMPKα siRNA prevented nuclear translocation of GAPDH in senescent HDFs. The stimulatory effect of AICAR and serum depletion on GAPDH nuclear translocation was reduced in AMPKα1/α2-knockout mouse embryonic fibroblasts. Overall, increased AMPK activity may play a role in the senescence-associated nuclear translocation of GAPDH.

GAPDH, Interferon γ, and Nitric Oxide: Inhibitors of Coronaviruses

As the COVID-19 pandemic finishes its second year, progress has been made against SARS-CoV-2 with vaccine candidates showing efficacy against this latest coronavirus strain. However, this pandemic presents a unique opportunity to investigate anti-viral therapies given the likely probability of another outbreak. One possible (and perhaps unlikely) therapeutic target could be GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Studies have show that downregulation of GAPDH leads to a decrease in interferon gamma (IFNγ production (which is an important cytokine response against coronaviruses and viruses in general). In this light, the previous coronavirus strain (SARS-CoV) has actually been shown to downregulate GAPDH. Although perhaps better known for its role in glycolysis, GAPDH also plays a role in gene expression of a varied set of genes by binding to their mRNA to affect stability and thereby translation Moreover, GAPDH is also upregulated by nitric oxide (NO), an inhibitor against both SARS-CoV and SARS-CoV-2. Additionally, GAPDH has also been shown to be a negative transcriptional regulator of AT1R (angiotensin II receptor 1), which has been shown to bind ACE2 for eventual endocytosis of the complex implicating GAPDH's potential role in the kinetics of coronavirus entry as well in downstream inflammatory signaling resulting from AT1R activation. Lastly, another important role for GAPDH is its requirement in the assembly of the GAIT complex that is responsible for termination of translation of IFNγ-responsive genes that would be critical for the resolution of any inflammatory response. These observations would imply that sufficient levels of GAPDH are needed for immune responses to function properly during a coronaviral infection. By examining different coronavirus studies, this review explores GAPDH's role as an inhibitor of coronaviruses (at the viral transcriptional level and also as a modulator of gene expression related to inflammation), and its signal transduction links to the IFNγ and NO pathways.

Characteristics and functions of glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation during controlled aging of elm and Arabidopsis seeds.

Seed aging is the gradual decline in seed vigor, during which programmed cell death (PCD) occurs. The functions of nitric oxide (NO) are exerted through protein S-nitrosylation, a reversible post-translational modification. During seed aging, more than 80 proteins are S-nitrosylated, but the particular role of individual proteins is unknown. Here, we showed that the S-nitrosylation level of glyceraldehyde 3-phosphate dehydrogenase (UpGAPDH) in elm (Ulmus pumila L.) seeds increased after controlled deterioration treatment. UpGAPDH was S-nitrosylated at Cys154 during S-nitrosoglutathione (GSNO) treatment, and its oligomerization was triggered both in vitro and in elm seeds. Interestingly, UpGAPDH interacted with the mitochondrial voltage-dependent anion channel in an S-nitrosylation-dependent way. Some UpGAPDH-green fluorescent protein in Arabidopsis protoplasts co-localized with mitochondria during the GSNO treatment, while the S-nitrosylation-defective UpGAPDH C154S-GFP protein did not. Seeds of oxUpGAPDH lines showed cell death and lost seed vigor rapidly during controlled deterioration treatment-triggered seed aging, while those overexpressing S-nitrosylation-defective UpGAPDH-Cys154 did not. Our results suggest that S-nitrosylation of UpGAPDH may accelerate cell death and seed deterioration during controlled deterioration treatment. These results provide new insights into the effects of UpGAPDH S-nitrosylation on protein interactions and seed aging.

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.

Glucose-dependent aerobic glycolysis contributes to recruiting viral components into HIV-1 particles to maintain infectivity

The cellular environment affects optimal viral replication because viruses cannot replicate without their host cells. In particular, metabolic resources such as carbohydrates, lipids, and ATP are crucial for viral replication, which is sensitive to cellular metabolism. Intriguingly, recent studies have demonstrated that human immunodeficiency virus type 1 (HIV-1) infection induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis in CD4+ T cells to produce the virus efficiently. However, the importance of aerobic glycolysis in maintaining the quality of viral components and viral infectivity has not yet been fully investigated. Here, we show that aerobic glycolysis is necessary not only to override the inhibitory effect of virion-incorporated glycolytic enzymes, but also to maintain the enzymatic activity of reverse transcriptase and the adequate packaging of envelope proteins into HIV-1 particles. To investigate the effect of metabolic remodeling on the phenotypic properties of HIV-1 produced by infected cells, we replaced glucose with galactose in the culture medium because the cells grown in galactose-containing medium are forced to carry out oxidative metabolism instead of aerobic glycolysis. We found that the packaging levels of glyceraldehyde 3-phosphate dehydrogenase, alpha-enolase and pyruvate kinase muscle type 2, which decrease HIV-1 infectivity by packaging into viral particles, are increased in progeny viruses produced by the cells grown in galactose-containing medium. Furthermore, we found that the entry and reverse transcription efficiency of the progeny viruses were reduced, which was caused by a decrease in the enzymatic activity of reverse transcriptase in the viral particles and a decrease in the packaging levels of envelope proteins and reverse transcriptase. These results indicate that the aerobic glycolysis environment in HIV-1-infected cells may contribute to the quality control of viruses.

Extracellular complex of beta‐amyloid with glyceraldehyde‐3‐phosphate dehydrogenase contributes to neurodegeneration in Alzheimer’s disease

AbstractBackgroundThe discovery of migrating protein complexes armed with β‐amyloid (Aβ) and acting as the driving force of Alzheimer’s disease (AD) mechanism led us to suggest that besides the pathogenic peptide some other proteins may cooperate with the latter and enhance its cytotoxicity and intercellular propagation. One of such proteins was suggested to be glyceraldehyde‐3‐phosphatedehydrogenase (GAPDH), enzyme that in normal conditions maintains cellular glycolysis and under action of oxidative stress is converted to monomers and dimers and forms tight, insoluble complexes with aggregation‐prone polypeptides.MethodCSF samples were collected from patients of both genders. The cohort comprised four patient groups: (1) mild cognitive impairment (MCI) n = 21; (2) mild AD‐type dementia (‘Mild’), n = 58; (3) moderate AD‐type dementia (‘Moderate’) n = 57; and (4) severe AD‐type dementia (‘Severe’), n = 56. The diagnosis was established according to the NINCDS‐ARDRA criteria for AD and the Petersen criteria for MCI. Patients were assigned to severity subgroups based on their scores on the Mini‐Mental State Examination (MMSE). Two animal models of AD were used in this study: (1) “chemical” model in rats and (2) transgenic 5XFAD mice.ResultGAPDH is present in cerebrospinal fluid samples of AD patients as covalent complex with Aβ, and the heavier the stage of the disease, the higher is the level of such complexes. The increase of constitutional GAPDH level in AD animal models results in aggravation of cognitive and brain deficits linked to AD progression.ConclusionThe findings imply a potential role of GAPDH as biomarker and prerequisite of massive neuronal death on the late stages of Alzheimer’s disease Supported by RSF grant #19‐74‐30007.

Evaluation of silent information regulator T (SIRT) 1 and Forkhead Box O (FOXO) transcription factor 1 and 3a genes in glaucoma.

Analysis of the reactive oxygen species (ROS)-detoxifying biomarkers may elucidate the mitochondrial dysfunction in glaucoma pathogenesis. Therefore, we purposed to investigate the effects of ROS-detoxifying molecules including Silent Information Regulator T1 (SIRT1) and Forkhead Box O 1 (FOXO1) and 3a (FOXO3a) transcription factors in patients with glaucoma. Our analyses included 20 eyes from patients with primary open-angle glaucoma (POAG) and 20 eyes from patients with pseudoexfoliation glaucoma (PXG) who were scheduled for trabeculectomy. After extraction of total RNA from trabecular meshwork tissue, we compared the levels of SIRT1, FOXO1and FOXO3a genes in the oxidative pathway with the level of glyceraldehyde-3 phosphate dehydrogenase (GAPDH), the reference gene, using real-time polymerase chain reaction. Relative gene expression was calculated using the threshold cycle (2-ΔΔCT) method. We observed similarly reduced expression levels of SIRT1, FOXO1, and FOXO3a genes versus GAPDH among patient groups (p = 0.40; p = 0.56; p = 0.35, respectively). This is the first study to identify the role of SIRT1 and FOXOs in human TM with glaucoma. Relative expression levels of SIRT1, FOXO1, and FOXO3a genes versus a control gene (GAPDH) were decreased in POAG and PXG groups. Our results show that SIRT1and FOXOs (1-3a) deserve special attention in the pathogenesis of glaucoma.

Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1Δ Mutant.

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Bioenergetic signature as a target of zinc oxide nanoparticles in Ehrlich ascitic carcinoma-bearing mice.

The current study aims to evaluate the modulatory effect of zinc oxide nanoparticles (ZnO NPs) on the bioenergetic signature biomarkers in the Ehrlich ascitic carcinoma (EAC) model. To achieve this goal, 90 female albino mice were included in this study and were divided into six equal groups (n =15 per group): saline-treated group, ZnO NP-treated, EACs-bearing mice, and three groups of EACs-bearing mice treated with ZnO NPs at a dose of 20 mg/kg every other day, 10 mg/kg every other day, 10 mg/kg every day, respectively, for 14 days. The tissues from treated groups and control groups were homogenized and used for the assay of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and F1 beta subunit of adenosine triphosphate (ATP) synthase levels, as well as the determination of lactate level. The survival time of mice was improved in all ZnO NP-treated groups, especially in EACs-bearing mice treated with ZnO NPs at a dose of 10 mg/kg every other day. This improvement was associated with an increased F1 beta subunit of ATP synthase level and a decreased GAPDH level. Also, the lactate level was significantly decreased in all treated groups when compared with the untreated group. The overall effect was the increased bioenergetic signature as compared with EC.These results implied that ZnO NPs have a significant efficacy against cancer cells and they significantly increased the bioenergetic signature.

Association between carbonyl stress markers and the risk of acute coronary syndrome in patients with type 2 diabetes mellitus–A pilot study

Background and aimsCarbonyl stress is one of the mechanisms responsible for diabetes and its complications. The study was planned to examine the relationship between carbonyl stress markers and the risk of acute coronary syndrome (ACS) in patients with type 2 diabetes mellitus (T2DM). MethodsForty T2DM patients with ACS and forty T2DM patients without ACS participated in this cross-sectional pilot study. Routine biochemical investigations, creatine kinase-total (CK-T), and creatine kinase-MB (CK-MB) levels were estimated. Serum carbonyl stress markers were analysed by enzyme-linked immunosorbent assay. Binary logistics regression was done to determine the predictive value of carbonyl stress markers for ACS. ResultsFasting plasma glucose, serum total methylglyoxal (MG), methylglyoxal derived hydroimidazolones-1 (MG-H1), and Nε-carboxymethyl-lysine (CML) levels were significantly higher in T2DM patients with ACS than in those without ACS. Serum glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glyoxalase-1 (GLO1) levels were significantly lower in T2DM patients with ACS than in those without ACS. Fasting plasma glucose level was significantly positively correlated with serum MG (r = 0.441, P < 0.001), CML (r = 0.649, P < 0.001), MG-H1 (r = 0.725, P < 0.001), and negatively correlated with serum GAPDH (r = – 0.268, P = 0.012) and GLO1 (r = – 0.634, P = 0.016). Receiver operating characteristic curve analysis showed that serum GAPDH and GLO1 could predict the risk of ACS in T2DM patients. ConclusionThese findings revealed that high carbonyl stress due to lower levels of GAPDH and GLO1 may predispose patients with T2DM for more risk of ACS.

An investigation of the correlation between the S-glutathionylated GAPDH levels in blood and Alzheimer's disease progression.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by two aggregates, namely, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein (tau-p), which are released into the blood in a very small amount and cannot be easily detected. An increasing number of recent studies have suggested that S-glutathionylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is highly correlated with Aβ in patients with AD and that S-glutathionylated GAPDH plays a role as a proapoptotic factor in AD. We found that S-glutathionylated GAPDH is abundant in the blood of AD patients, which is unusual because S-glutathionylated GAPDH cannot exist in the blood under normal conditions. The aim of this study was to further explore the correlation between the S-glutathionylated GAPDH levels in blood plasma and AD progression. As controls, we recruited 191 people without AD, which included 111 healthy individuals and 37 patients with depression and insomnia, in the psychosomatic clinic. Moreover, 47 patients with AD (aged 40–89 years) were recruited at the neurology clinic. The blood S-glutathionylated GAPDH levels in the AD patients were significantly (p < 0.001) higher (752.7 ± 301.7 ng/dL) than those in the controls (59.92 ± 122.4 ng/dL), irrespective of gender and age. For AD diagnosis, the criterion blood S-glutathionylated GAPDH level > 251.62 ng/dL exhibited 95.74% sensitivity and 92.67% specificity. In fact, the individuals aged 70–89 years, namely, 37 patients from the psychosomatic clinic and 42 healthy individuals, showed significant blood S-glutathionylated GAPDH levels (230.5 ± 79.3 and 8.05 ± 20.51 ng/dL, respectively). This finding might indicate neurodegenerative AD progression in psychosomatic patients and suggests that the degree of neuronal apoptosis during AD progression might be sensitively evaluated based on the level of S-glutathionylated GAPDH in blood.

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).

Evaluation of the Housekeeping Genes; β‐Actin, Glyceraldehyde‐3‐Phosphate‐Dehydrogenase, and 18S rRNA for Normalization in Real‐Time Polymerase Chain Reaction Analysis of Gene Expression in Human Adipose Tissue

Background: Several studies suggested that beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA are expressed constitutively and contribute to the fundamental reference actions essential for cell viability and maintenance. However, there are inconsistency in this regard. Hence, we aimed to evaluate the accuracy of these three potential reference genes for Real‐Time quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) application for normalization in two types of human adipose tissues. Materials and Methods: Subcutaneous and visceral fat tissues were derived from 19 healthy and 20 obese subjects and RT-qPCR was applied to determine the expression levels of beta-actin, GAPDH, and18S rRNA. Results: The gene expression level of beta-actin, GAPDH, and 18S rRNA was essentially the same in the subcutaneous and visceral fat tissues of all participants (P>0.05). Hence, all considered housekeeping genes displayed high expression stability and the analysis revealed that normalization to all of these three housekeeping genes gave a result that satisfactorily reflected the acceptable mRNA expression levels in adipose tissues. Conclusion: Collectively, our findings suggest of beta-actin, GAPDH, and18S rRNA as reference genes applicable in human adipose tissue in the context of obesity.