Glyceraldehyde-3-phosphate Dehydrogenase Knockdown Research Articles

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after −1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after −1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at −1 V compared with the same at ±2–3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.

Glyceraldehyde-3-phosphate dehydrogenase regulates cyclooxygenase-2 expression by targeting mRNA stability

Cyclooxygenase (COX)-2 is an inducible inflammatory protein whose expression is partially regulated at the post-transcriptional level. We investigated whether glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds to the AU-rich element (ARE) of COX-2 mRNA for its degradation. Knockdown of GAPDH in hepa1c1c7 cells significantly enhanced COX-2 expressions. Recombinant GAPDH bound to the COX-2 ARE within the first 60 nucleotides of the 3′-UTR via the NAD+ binding domain. Interestingly, a C151S GAPDH mutant retained binding activity. Confocal microscopy observation revealed that LPS exposure reduced the localization of GAPDH in nuclei. Our results indicate that GAPDH negatively regulates COX-2 by binding to its ARE.

Targeting the glycolytic pathway in cancer cells: glycolytic and regulatory functions of GAPDH

Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) is a pivotal enzyme of the glycolytic pathway, and a regulatory protein in several cellular networks. Knockdown of GAPDH induces senescence in human cancer cells A549, as revealed by senescence biomarkers SA‐â‐galactosidase and DEC1. To assess the potential role of GAPDH as a molecular target within the cancer energy metabolism network, we (1) characterized structural elements of GAPDH polypeptide pertinent to its intranuclear functions, and (2) evaluated sensitivity of GAPDH‐depleted cells against a panel of chemotherapeutic agents. We performed site‐mutagenesis of the functional domains in GAPDH, and estimated the enzymatic properties, intracellular distribution, and intranuclear mobility of GAPDH mutated variants. The fluorescence recovery after photobleaching experiments revealed that the substitution of Thr and Ser by non‐phosphorylated amino acid residues significantly increased intranuclear mobility of GAPDH. Our results indicate that GAPDH phosphorylation is important both for GAPDH enzymatic activity, and interactions with intranuclear components. Identification of intranuclear functional partners of GAPDH is expected to define targets for development of novel antiproliferative agents.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-β-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

Glyceraldehyde-3-phosphate dehydrogenase: activity inhibition and protein overexpression in rotenone models for Parkinson's disease

Rotenone, a widely used pesticide and an environmental risk factor for Parkinson's disease (PD), induces nigrostriatal injury, Lewy body-like inclusions, and Parkinsonian symptoms in rat models for PD. Our previous data indicated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overexpression and glycolytic inhibition were co-current in rotenone-induced PC12 (rat adrenal pheochromocytoma cells) cell death. However, whether GAPDH overexpression plays any role in dopaminergic neurodegeneration in vivo remains unknown. In this study, we have found that GAPDH overexpression and GAPDH-positive Lewy body-like aggregates in nigral dopaminergic neurons while nigral GAPDH glycolytic activity decreases in rotenone-based PD animal models. Furthermore, GAPDH knockdown reduces rotenone toxicity significantly in PC12. These in vitro and in vivo data suggest that GAPDH contributes to the pathogenesis of Parkinson's disease, possibly representing a new molecular target for neuroprotective strategies and alternative therapies for PD.

Energy metabolic block induces accelerated senescence in human cancer cells

Glycolytic pathway is an essential source of energy in cancer cells; therefore, it can be selectively targeted by anticancer pharmacotherapy. The pivotal enzyme in this pathway is glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) which executes both enzymatic and regulatory functions. We evaluated the role of GAPDH in the control of energy homeostasis in isogenic (p53+/+ and p53−/−) human carcinoma cells by using RNA interference, site mutagenesis, and confocal microscopy. Knockdown of GAPDH induced accelerated senescence of cancer cells via p53‐regulated pathway, in the absence of DNA damage. Depletion of GAPDH by short interfering RNA decreased protein level, GAPDH enzymatic activity, and ATP. In response to reduced ATP, AMPK pathway was activated leading to p53 stabilization, and cell proliferation arrest. GAPDH‐depleted cells acquired senescence phenotype revealed by changes in morphology and senescence‐associated markers. Rescue experiments aimed to reinstate energy metabolism (by adding the downstream metabolite pyruvate), or GAPDH level partially restored ATP level but not cell proliferation suggesting that energy crisis by GAPDH depletion may not be the only cause of accelerated senescence. These results indicate that accelerated senescence induced by GAPDH depletion may provide a strategy to control proliferation of cancer cells.This work was supported in part by NCI grant R01 CA104729.

Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo

Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room. Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mm Hg hyperbaric pressure, or (iii) 120 mm Hg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry. Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared with hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods. Endothelial gene silencing is possible within the time frame and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease.

Efficient Gene Silencing in Lungs and Liver Using Imidazole-Modified Chitosan As a Nanocarrier for Small Interfering RNA

Despite high specificity and potency, small interfering RNA (siRNA)-based therapeutics have been limited by their poor biostability and intracellular penetration. Thus, effective nanocarriers that can protect and efficiently deliver siRNA to target cells in vivo are needed. Here we report on the efficiency of imidazole-modified chitosan (chitosan-imidazole-4-acetic acid [IAA])-siRNA nanoparticles to mediate gene silencing after administration via either intravenous (i.v.) or intranasal (i.n.) routes. Poly(ethylene glycol) (PEG)ylated nanoparticles for i.v. delivery demonstrated significant knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme in both lung and liver at as low as 1 mg/kg siRNA dose. In addition, the efficient, dose-dependent silencing of apolipoprotein B in the liver was also shown. For i.n. delivery, significant silencing of GAPDH protein expression was seen in the lungs with only 0.5 mg/kg/day siRNA delivered over 3 consecutive days. In summary, imidazole-modified chitosan-IAA nanoparticles are potentially effective carriers for siRNA delivery.

GAPDH binds GLUT4 reciprocally to hexokinase-II and regulates glucose transport activity

Dietary glucose is taken up by skeletal muscle through GLUT4 (glucose transporter 4). We recently identified by MS proteins displaying insulin-dependent co-precipitation with Myc-tagged GLUT4 from L6 myotubes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HKII (hexokinase-II). In the present paper we explored whether GAPDH and HKII interact directly with cytoplasmic regions of GLUT4 and their possible inter-relationship. Endogenous and recombinant GAPDH and HKII bound to a chimeric protein linearly encoding all three cytosolic domains of GLUT4 [GST (glutathione-transferase)-GLUT4-cyto]. Both proteins bound to a lesser extent the middle cytosolic loop but not individual N- or C-terminal domains of GLUT4. Purified GAPDH and HKII competed for binding to GST-GLUT4-cyto; ATP increased GAPDH binding and decreased HKII binding to this construct. The physiological significance of the GAPDH-GLUT4 interaction was explored by siRNA (small interfering RNA)-mediated GAPDH knockdown. Reducing GAPDH expression by 70% increased HKII co-precipitation with GLUT4-Myc from L6 cell lysates. GAPDH knockdown had no effect on surface-exposed GLUT4-Myc in basal or insulin-stimulated cells, but markedly and selectively diminished insulin-stimulated 3-O-methyl glucose uptake and GLUT4-Myc photolabelling with ATB-BMPA {2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-propylamine}, suggesting that the exofacial glucose-binding site was inaccessible. The results show that GAPDH and HKII reciprocally interact with GLUT4 and suggest that these interactions regulate GLUT4 intrinsic activity in response to insulin.

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.

Interaction of glyceraldehyde‐3‐phosphate dehydrogenase in the light‐induced rod alpha‐transducin translocation

The light-dependent subcellular translocation of rod alpha-transducin (GNAT-1, or rod Talpha) has been well documented. In dark-adapted animals, rod Talpha (rTalpha) is predominantly located in the rod outer segment (ROS) and translocates into the rod inner segment (RIS) upon exposure to the light. Neither the molecular participants nor the mechanism(s) involved in this protein trafficking are known. We hypothesized that other proteins must interact with rTalpha to affect the translocations. Using the MBP-rTalpha fusion pulldown assay, the yeast two-hybrid assay and the co-immunoprecipitation assay, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rTalpha as interacting proteins. Immunoprecipitation also showed beta-actin associates with rTalpha in the dark but not in the light. To further investigate the involvement of GAPDH in light-induced rod Talpha translocation, GAPDH mRNA was knocked down in vivo by transient expression of siRNAs in rat photoreceptor cells. Under completely dark- and light-adapted conditions, the translocation of rTalpha was not significantly different within the 'GAPDH knock-down photoreceptor cells' compared to the non-transfected control cells. However, under partial dark-adaptation, rTalpha translocated more slowly in the 'GAPDH knock-down cells' supporting the conclusion that GAPDH is involved in rTalpha translocation from the RIS to the ROS during dark adaptation.

Glyceraldehyde-3-phosphate Dehydrogenase Enhances Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) functions as a transcriptional factor for genes involved in proliferation and differentiation of normal and cancerous prostate cells. Coactivators that bind to AR are required for maximal androgen action. Here we report that increasing the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a prostate cancer cell line by as little as 1.8-fold enhances transcriptional activity of AR (but not the transcriptional activity of glucocorticoid receptor or estrogen receptor alpha) in a ligand-dependent manner and results in an increased expression of prostate-specific antigen. Small interference RNA-mediated knockdown of GAPDH significantly attenuated ligand-activated AR transactivation. Immunoprecipitation analysis revealed the presence of an endogenous protein complex containing GAPDH and AR in both the cytoplasm and nucleus. Addition of a nuclear localization signal (NLS) to GAPDH (GAPDH-NLS) completely abolished the ability of GAPDH to transactivate AR. Neither wild-type GAPDH nor GAPDH-NLS enhanced transcriptional activity of mutant AR (AR Delta C-Nuc) that is a constitutively active form of AR in the nucleus, even though GAPDH-NLS formed a complex with wild-type AR or AR Delta C-Nuc. AR transactivation was enhanced by a mutant GAPDH lacking dehydrogenase activity. GAPDH enhanced the transcriptional activity of AR(T875A) activated by an antagonist such as hydroxyflutamide or cyproterone acetate. These results indicate that GAPDH functions as a coactivator with high selectivity for AR and enhances AR transactivation independent of its glycolytic activity. Further, these data suggest that formation of a GAPDH.AR complex in the cytoplasm rather than nucleus is essential for GAPDH to enhance AR transactivation.

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, APOPTOSIS, AND NEURODEGENERATIVE DISEASES

Increasing evidence supports the notion that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with multiple functions, including its surprising role in apoptosis. GAPDH is overexpressed and accumulates in the nucleus during apoptosis induced by a variety of insults in diverse cell types. Knockdown of GAPDH using an antisense strategy demonstrates its involvement in the apoptotic cascade in which GAPDH nuclear translocation appears essential. Knowledge concerning the mechanisms underlying GAPDH nuclear translocation and subsequent cell death is growing. Additional evidence suggests that GAPDH may be an intracellular sensor of oxidative stress during early apoptosis. Abnormal expression, nuclear accumulation, changes in physical properties, and loss of glycolytic activity of GAPDH have been found in cellular and transgenic models as well as postmortem tissues of several neurodegenerative diseases. The interaction of GAPDH with disease-related proteins as well as drugs used to treat these diseases suggests that it is a potential molecular target for drug development.