Overexpression Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage

BackgroundEarly brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. MethodsThe rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. ResultsWe observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. ConclusionThis study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.

Effects of simultaneous Rubisco, glyceraldehyde-3-phosphate dehydrogenase, and triosephosphate isomerase overexpression on photosynthesis in rice

ABSTRACT Rubisco overexpression does not lead to substantial photosynthesis improvement. One of the possible causes is the over-accumulation of 3-phosphoglycerate in the Calvin–Benson cycle. We have previously suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI), involved in 3-phosphoglycerate metabolism, have control over the Calvin–Benson cycle metabolism and photosynthesis. In the present study, Rubisco, GADPH, and TPI were simultaneously overexpressed in rice plants through crossing to improve photosynthesis and its N use efficiency. The percentage of N allocated to Rubisco and GAPDH and TPI levels in the transgenic plant increased 1.1-, 4.9-, and 2.1-fold, respectively, compared to those in wild-type plants, showing overexpression of these enzymes. However, there were no marked increases in CO2 assimilation rates at different CO2 levels and capacities of Rubisco carboxylation, regeneration of Rubisco substrate, and triose phosphate use calculated using the CO2 assimilation rates in the transgenic plants. The ratios of these photosynthetic capacities to total leaf-N level tended to be slightly lower in the transgenic plants than those in the wild-type plants, implying a marginal decrease in photosynthetic N use efficiency. In the transgenic rice plants substantially overexpressing GAPDH and TPI, photosynthesis at elevated CO2 levels, which is assumed to be limited factors other than Rubisco, was only slightly improved. Therefore, it is suggested that simultaneous Rubisco, GAPDH, and TPI overexpression do not improve photosynthesis and its N use efficiency, as GAPDH and TPI overexpression do not improve the 3-phosphoglycerate metabolism sufficiently to alleviate its over-accumulation caused by Rubisco overexpression.

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.

Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes.

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes μL-1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

CASC3 Biomolecular Condensates Restrict Turnip Crinkle Virus by Limiting Host Factor Availability

The exon-junction complex (EJC) plays a role in post-transcriptional gene regulation and exerts antiviral activity towards several positive-strand RNA viruses. However, the spectrum of RNA viruses that are targeted by the EJC or the underlying mechanisms are not well understood. EJC components from Arabidopsis thaliana were screened for antiviral activity towards Turnip crinkle virus (TCV, Tombusviridae). Overexpression of the accessory EJC component CASC3 inhibited TCV accumulation > 10-fold in Nicotiana benthamiana while knock-down of endogenous CASC3 resulted in a > 4-fold increase in TCV accumulation. CASC3 forms cytoplasmic condensates and deletion of the conserved SELOR domain reduced condensate size 7-fold and significantly decreased antiviral activity towards TCV. Mass spectrometry of CASC3 complexes did not identify endogenous stress granule or P-body markers and CASC3 failed to co-localize with an aggresome-specific dye suggesting that CASC3 condensates are distinct from well-established membraneless compartments. Mass spectrometry and bimolecular fluorescence complementation assays revealed that CASC3 sequesters Heat shock protein 70 (Hsp70-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two host factors with roles in tombusvirus replication. Overexpression of Hsp70-1 or GAPDH reduced the antiviral activity of CASC3 2.1-fold and 2.8-fold, respectively, and suggests that CASC3 inhibits TCV by limiting host factor availability. Unrelated Tobacco mosaic virus (TMV) also depends on Hsp70-1 and CASC3 overexpression restricted TMV accumulation 4-fold and demonstrates that CASC3 antiviral activity is not TCV-specific. Like CASC3, Auxin response factor 19 (ARF19) forms poorly dynamic condensates but ARF19 overexpression failed to inhibit TCV accumulation and suggests that CASC3 has antiviral activities that are not ubiquitous among cytoplasmic condensates.

GAPDH: A common housekeeping gene with an oncogenic role in pan-cancer

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is one of the most prominent housekeeping proteins and is widely used as an internal control in some semi-quantitative assays. In addition to glycolysis, GAPDH is involved in several cancer-related biological processes and has been reported to be commonly dysregulated in multiple cancer types. Therefore, its role in the physiological process of cancer needs to be urgently elucidated. Pan-cancer analysis indicated that GAPDH is ubiquitously highly expressed in most cancer types, and that patients with a high GAPDH expression of in tumor tissues have a poor prognosis. The concordance of GAPDH expression in tumors with the infiltration of immune cells and immune checkpoints implies a certain association between GAPDH and the tumor microenvironment as well as tumor development. Gene Set Enrichment Analysis revealed that GAPDH may contribute to multiple important cancer-related pathways and biological processes. Multi-omics analysis and in vitro cell experiments revealed that GAPDH overexpression is regulated by DNA copy number amplification and promoter methylation modification. Importantly, a transcription factor, forkhead box M1 (FOXM1), which is capable of regulating GAPDH expression, was also identified and was confirmed to be an oncogene and ubiquitously highly expressed in multiple cancer types. Semi-quantitative chromatin immunoprecipitation, quantitative PCR, and dual-luciferase assays showed that FOXM1 mainly binds to the promoter region of GAPDH in two cancer cell lines. The present findings revealed the implication of GAPDH in tumor development, thus bringing attention to this important molecule and casting doubts on its role as an internal reference gene in cancer studies.

Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis, has evolved mechanisms to evade host defenses and persist within host cells. Host-directed therapies against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of purinergic receptor 7, the receptor previously reported for this peptide, remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft-dependent process. We also discovered a role for GAPDH in LL-37-mediated autophagy induction and clearance of intracellular pathogens. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37-mediated autophagy which was rescued by GAPDH overexpression. This process was dependent on intracellular calcium and p38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggest a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden.

TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression

Increased expression of transketolase (TKT) and its isoform transketolase-like-1 (TKTL1) has been related to the malignant leukemia phenotype through promoting an increase in the non-oxidative branch of the pentose phosphate pathway (PPP). Recently, it has also been described that TKTL1 can have a role in survival under hypoxic conditions and in the acquisition of radio resistance. However, TKTL1’s role in triggering metabolic reprogramming under hypoxia in leukemia cells has never been characterized. Using THP-1 AML cells, and by combining metabolomics and transcriptomics techniques, we characterized the impact of TKTL1 knockdown on the metabolic reprogramming triggered by hypoxia. Results demonstrated that TKTL1 knockdown results in a decrease in TKT, glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities and impairs the hypoxia-induced overexpression of G6PD and GAPDH, all having significant impacts on the redox capacity of NADPH- and NADH-related cells. Moreover, TKTL1 knockdown impedes hypoxia-induced transcription of genes encoding key enzymes and transporters involved in glucose, PPP and amino acid metabolism, rendering cells unable to switch to enhanced glycolysis under hypoxia. Altogether, our results show that TKTL1 plays a key role in the metabolic adaptation to hypoxia in THP-1 AML cells through modulation of G6PD and GAPDH activities, both regulating glucose/glutamine consumption and the transcriptomic overexpression of key players of PPP, glucose and amino acids metabolism.

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract

ABSTRACT Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10–28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis

AimsS-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. ResultsExposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10–15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. ConclusionsAbundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Sperm-Specific Glycolysis Enzyme Glyceraldehyde-3-Phosphate Dehydrogenase Regulated by Transcription Factor SOX10 to Promote Uveal Melanoma Tumorigenesis.

Melanoma cells exhibit increased aerobic glycolysis, which represents a major biochemical alteration associated with malignant transformation; thus, glycolytic enzymes could be exploited to selectively target cancer cells in cancer therapy. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) switches glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate by coupling with the reduction of NAD+ to NADH. Here, we demonstrated that GAPDHS displays significantly higher expression in uveal melanoma (UM) than in normal controls. Functionally, the knockdown of GAPDHS in UM cell lines hindered glycolysis by decreasing glucose uptake, lactate production, adenosine triphosphate (ATP) generation, cell growth and proliferation; conversely, overexpression of GAPDHS promoted glycolysis, cell growth and proliferation. Furthermore, we identified that SOX10 knockdown reduced the activation of GAPDHS, leading to an attenuated malignant phenotype, and that SOX10 overexpression promoted the activation of GAPDHS, leading to an enhanced malignant phenotype. Mechanistically, SOX10 exerted its function by binding to the promoter of GAPDHS to regulate its expression. Importantly, SOX10 abrogation suppressed in vivo tumor growth and proliferation. Collectively, the results reveal that GAPDHS, which is regulated by SOX10, controls glycolysis and contributes to UM tumorigenesis, highlighting its potential as a therapeutic target.

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.

Extracellular GAPDH Promotes Alzheimer Disease Progression by Enhancing Amyloid-β Aggregation and Cytotoxicity.

Neuronal cell death at late stages of Alzheimer’s disease (AD) causes the release of cytosolic proteins. One of the most abundant such proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forms stable aggregates with extracellular amyloid-β (Aβ). We detect these aggregates in cerebrospinal fluid (CSF) from AD patients at levels directly proportional to the progressive stages of AD. We found that GAPDH forms a covalent bond with Q15 of Aβ that is mediated by transglutaminase (tTG). The Q15A substitution weakens the interaction between Aβ and GAPDH and reduces Aβ-GAPDH cytotoxicity. Lentivirus-driven GAPDH overexpression in two AD animal models increased the level of apoptosis of hippocampal cells, neural degeneration, and cognitive dysfunction. In contrast, in vivo knockdown of GAPDH reversed these pathogenic abnormalities suggesting a pivotal role of GAPDH in Aβ-stimulated neurodegeneration. CSF from animals with enhanced GAPDH expression demonstrates increased cytotoxicity in vitro. Furthermore, RX-624, a specific GAPDH small molecular ligand reduced accumulation of Aβ aggregates and reversed memory deficit in AD transgenic mice. These findings argue that extracellular GAPDH compromises Aβ clearance and accelerates neurodegeneration, and, thus, is a promising pharmacological target for AD.

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.

Prognostic implications of markers of the metabolic phenotype in human cutaneous melanoma.

Reprogramming of energy metabolism to enhanced aerobic glycolysis has been defined as a hallmark of cancer. To investigate the role of the mitochondrial proteins, β-subunit of the H+ -ATP synthase (β-F1-ATPase), and heat-shock protein 60 (HSP60), and the glycolytic markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as the bioenergetic cellular (BEC) index, in melanoma progression. The expression of energy metabolism proteins was assessed on a set of different melanoma cells representing the natural biological history of the disease: primary cultures of melanocytes, radial (WM35) and vertical (WM278) growth phases, and poorly (C81-61-PA) and highly (C8161-HA) aggressive melanoma cells. Cohorts of 63 melanocytic naevi, 55 primary melanomas and 35 metastases were used; and 113 primary melanoma and 33 metastases were used for validation. The BEC index was significantly reduced in melanoma cells and correlated with their aggressive characteristics. Overexpression of HSP60, GAPDH and PKM2 was detected in melanoma human samples compared with naevi, showing a gradient of increased expression from radial growth phase to metastatic melanoma. The BEC index was also significantly reduced in melanoma samples and correlated with worse overall and disease-free survival; the multivariate Cox analysis showed that the BEC index (hazard ratio 0·64; 95% confidence interval 0·4-1·2) is an independent predictor for overall survival. A profound alteration in the mitochondrial and glycolytic proteins and in the BEC index occurs in the progression of melanoma, which correlates with worse outcome, supporting that the alteration of the metabolic phenotype is crucial in melanoma transformation.

Effect of GAPDS overexpression on high glucose-induced oxidative damage

The occurrence of infertility in diabetic patients is attributed to oxidative damage of peroxidized products. High glucose-induced mitochondrial oxidative stress and glycolytic enzyme inactivation is considered to be an important mediator for sperm dysfunction. In this study, we successfully constructed TM3-GAPDS stable strain and investigated the role of sperm specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) on high glucose-induced apoptosis in TM3 cells. High glucose decreased the protein expression of SOD2 and catalase, while the level of intracellular ROS and the apoptosis - related protein increased in TM3 cells. Furthermore, high glucose-induced oxidative stress and apoptosis were reversed by GAPDS overexpression or antioxidant treatment. In conclusion, our data suggest that GAPDS overexpression antagonize high glucose-induced apoptosis by controlling ROS accumulation in TM3 cells.

Glyceraldehyde-3-phosphate dehydrogenase promotes liver tumorigenesis by modulating phosphoglycerate dehydrogenase.

GAPDH increases histone methylation levels by up-regulating PHGDH, promoting diversion from glycolysis to serine biosynthesis, and consequently accelerating HCC development. (Hepatology 2017;66:631-645).