Translocation Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

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.

Nuclear translocation of GluA2/ GAPDH promotes neurotoxicity after pilocarpine-induced epilepsy

Recent studies have identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exerts multiple functions besides its role in energy metabolism. It can form a protein complex with GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), translocate into nucleus and confer neurotoxicity in a cerebral ischemia model. We postulate that GAPDH may also induce neurotoxicity by forming a coupling with GluA2 in pilocarpine-induced epileptic model, and disruption of the GluA2/GAPDH coupling can protect against neuronal injury. In this study, induced status epilepticus (SE) in rats by the systemic administration of pilocarpine, collected hippocampal tissues at different time points after SE, and assessed the relationship between GluA2/GAPDH coupling and neuronal apoptosis in SE rats. Then, we interrupted the GluA2/GAPDH coupling by a special interfering peptide and determined whether neuronal injury can be rescued and hippocampus-depended memory function can be improved. We also evaluated the concentrations of GAPDH in nuclear and cytoplasmatic proteins in SE group, non-SE group and after interruption of GluA2/GAPDH coupling, to verify the nuclear translocation of GAPDH in SE model. We found that the apoptosis of hippocampal neurons was most significant at 72 h after SE, which was also the peak time of GluA2/GAPDH coupling expression and GluA2 consumption. After interruption of GluA2/GAPDH coupling, the apoptosis and memorial function of hippocampal neurons were improved and nuclear translocation of GluA2/GAPDH coupling was reduced. In conclusion, GAPDH can be translocated into nucleus in the form of GluA2/GAPDH, which plays an important role in regulating pilocarpine-induced epilepsy via neurotoxicity pathway.

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.

Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

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.

Regulation of Endothelium-Reticulum-Stress-Mediated Apoptotic Cell Death by a Polymethoxylated Flavone, Nobiletin, Through the Inhibition of Nuclear Translocation of Glyceraldehyde 3-Phosphate Dehydrogenase in Retinal Müller Cells.

In the early stages of diabetic retinopathy (DR), subtle biochemical and functional alterations occur in Müller cells, which are one of the components of the blood–retinal barrier (BRB). Müller cells are the principal glia of the retina and have shown a strong involvement in the maintenance of homeostasis and the development of retinal tissue. Their functional abnormalities and eventual loss have been correlated with a decrease in the tight junctions between endothelial cells and a consequent breakdown of the BRB, leading to the development of DR. We demonstrated that the endothelium reticulum (ER) triggers Müller cell death and that nuclear accumulation of glyceraldehyde 3-phosphate dehydrogenase is closely associated with ER-induced Müller cell death. In addition, induction of ER stress in Müller cells increased vascular endothelial growth factor expression but decreased pigment-epithelium-derived factor (PEDF) expression in Müller cells. We found that nobiletin, a polymethoxylated flavone from citrus explants, exerts protective action against ER-stress-induced Müller cell death. In addition, nobiletin was found to augment PEDF expression in Müller cells, which may lead to the protection of BRB integrity. These results suggest that nobiletin can be an attractive candidate for the protection of the BRB from breakdown in DR.

Stk and Stp1 participate in Streptococcus suis serotype 2 pathogenesis by regulating capsule thickness and translocation of certain virulence factors

Eukaryotic-like serine/threonine protein kinase (eSTK) and phosphatase (eSTP) play multiple roles in pathogenesis of many Gram-positive bacteria. eSTK (Stk) and eSTP (Stp1) of Streptococcus suis serotype 2 (S. suis 2) have also been reported to be virulence-associated, but their roles and underlying mechanisms in S. suis 2 pathogenesis require further investigation. We constructed mutants of stk or stp1 deletion using the virulent S. suis 2 isolate 05ZYH33 as the parental strain. Both Δstk and Δstp1 mutants showed abnormal cell division shown as increased chain length. This might be due to regulation by Stk and Stp1 of the phosphorylation status of the bacterial division protein DivIVA. Both mutants showed increased adhesion but reduced invasion to epithelial and endothelial cells. The two mutants were more readily phagocytosed by murine RAW264.7 macrophages. Western blotting revealed that GAPDH (glyceraldehyde-3-phosphate dehydrogenase), an important adhesin of S. suis, was significantly increased in the surface-associated and secreted fractions of the two mutant strains. Because increased adhesion of the mutant strains Δstk and Δstp1 to endothelial cells could be significantly inhibited by anti-GAPDH serum, we suppose that aberrant translocation of GAPDH due to deletion of the stk or stp1 gene contributed to increased interaction with host cells. The Δstk mutant showed reduced survival in macrophages, while the Δstp1 mutant showed increased survival probably as a result of increased capsule thickness. Enhanced hemolytic activity of the Δstk mutant could be due to increased secretion of suilysin. Both mutants exhibited reduced survival in pig whole blood and attenuated virulence to mice. Taken together, these results suggest that Stk and Stp1 can modulate S. suis cell division by post-translational modification of DivIVA, and regulate translocation of certain virulence factors, thereby benefiting its pathogenicity by compromising its interactions with the host.

E3 ubiquitin ligase siah‑1 nuclear accumulation is critical for homocysteine‑induced impairment of C6 astroglioma cells.

Elevated plasma homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is an independent risk factor for neurodegenerative diseases. Hcy, even at a low concentration, can promote free radical formation and increase oxidative stress, leading to neuronal death, which may be an important mechanism underlying the pathogenesis of neurodegenerative diseases. Although several reports have indicated that the nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be involved in Hcy-induced apoptosis, the exact mechanism remains to be fully elucidated. The siah E3 ubiquitin protein ligase 1 (siah-1) gene was found to be critical for the translocation of GAPDH from the cytoplasm to the nucleus. In the present study, the role of siah-1 was investigated in the nuclear translocation of GAPDH in rat C6 astroglioma cells treated with Hcy. C6 cells were treated with various concentrations of Hcy for 48 h and the expression level of siah-1 was examined using reverse transcription-quantitative polymerase chain reaction and western blotting analysis. In addition, the subcellular localization of siah-1 and GAPDH and the interaction between these two factors were investigated by immunofluorescence staining and co-immunoprecipitation assay, respectively. The results showed that Hcy at a high concentration increased the expression of siah-1 and induced nuclear translocation of siah-1 and GAPDH. In addition, siah-1 knockdown by siah-1 small interfering RNA significantly decreased the Hcy-induced nuclear accumulation of GAPDH and inhibited the impairment of C6 cells. These findings suggest that siah-1 is involved in Hcy-induced cell damage by promoting the nuclear translocation of GAPDH.

Nuclear GAPDH is vital for hypoxia-induced hepatic stellate cell apoptosis and is indicative of aggressive hepatocellular carcinoma behavior.

Background/aim: Hepatic stellate cells (HSCs) are critical determinants of liver tumor behavior such as vascular invasion, cell proliferation and migration. The apoptosis of HSCs can inhibit tumor growth and contribute to repressing hepatocellular carcinoma (HCC) progression. Our study aims to investigate the impact of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on HSCs under hypoxic conditions and the association of nuclear GAPDH with HCC patient outcomes and tumor progression.Patients and methods: Following stable cell passage, 0.3% O2 was used to induce hypoxia. Cell proliferation and apoptosis were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays and flow cytometry, respectively. Proteins expression were detected by extracting nuclear and cytoplasmic proteins and performing Western blots. GAPDH nuclear translocation was blocked by the agent deprenyl. Immunohistochemical staining for GAPDH was investigated in 137 HCC tissue samples from our center. An analysis of the clinicopathological features, Kaplan-Meier analysis and Cox proportional hazards regression analysis were applied.Results: MTT assays and flow cytometry analyses showed that the nuclear accumulation of GAPDH led to the apoptotic death of HSCs, while blockade of this process with deprenyl significantly decreased apoptosis. Western blots revealed that deprenyl inhibited the nuclear translocation of GAPDH. An analysis of the immunohistochemical staining of HSCs in HCC tissue samples (137) revealed that nuclear GAPDH expression was significantly positively correlated with HIF-1α expression. Overall survival (OS) and time-to-recurrence (TTR) estimated by Kaplan-Meier analyses showed that patients with high HIF-1α or low nuclear GAPDH levels in HSCs had significantly poorer prognosis compared with patients with low HIF-1α or high nuclear GAPDH expression in HSCs. Moreover, patients with combined high HIF-1α/low nuclear GAPDH expression in HSCs had the worst prognosis. The Cox regression analysis revealed that the combination of nuclear GAPDH/HIF-1α expression in HSCs was an independent prognostic factor for OS and TTR in HCC patients.Conclusions: These findings provide a novel mechanism underlying the involvement of intranuclear GAPDH in hypoxia-induced HSCs apoptosis and a correlation between nuclear GAPDH levels and the clinical prognosis, which may prompt the development of a novel therapeutic strategy for HCC.

Inhibiting a spinal cord signaling pathway protects against ischemia injury in rats

ObjectiveThe aim of the study was to examine whether the cannabinoid agonist WIN55212-2 could attenuate ischemic spinal cord injury (SCI) in rats through inhibition of GAPDH/Siah1 signaling. MethodsMale Sprague-Dawley rats were distributed randomly into 5 groups: (1) sham group that received no aortic occlusion and injected intraperitoneally (i.p.) with vehicle control after reperfusion; (2) control group that received a 12-minute aortic occlusion and injected i.p. with vehicle control after reperfusion; (3) WIN55212-2 group (WIN) that received the aortic occlusion and injected i.p. with 1 mg/kg of WIN55212-2 after reperfusion; and (4) WIN55212-2 plus AM251 group and (5) WIN55212-2 plus AM630 group that received the same surgical operation as the WIN group, except that 1 mg/kg of AM251 or AM630 was injected i.p. 30 min before each dose of WIN55212-2 injection, respectively. Neurologic function was assessed 48 hours after reperfusion. Histopathologic examination was performed to determine the number of normal neurons in anterior spinal cord. Protein expression of active caspase-3, total caspase-3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), nuclear factor kappa light chain enhancer of activated B cells (NF-κB), Siah1, tumor necrosis factor α, and interleukin 1β were determined with Western blot and enzyme-linked immunosorbent assay; coimmunoprecipitation assays were also used to determine GAPDH/Siah1 complexing. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to determine neuronal apoptosis in the lumbar spinal cord. ResultsThe nuclear translocation of GAPDH and Siah1 in the spinal cord was initiated after ischemic spinal cord injury (SCI) along with the increased formation of GAPDH/Siah1 complexes. However, the activation of GAPDH/Siah1 was blocked by WIN. In addition, the treatment of WIN55212-2 promoted neuronal survival in the spinal cord, reduced apoptosis and inflammation, and improved neurologic scores. Furthermore, these beneficial effects of WIN55212-2 were abolished by the combined treatment of the CB2 antagonist AM630, but not the CB1 antagonist AM251. ConclusionsOur findings reveal GAPDH/Siah1 signaling cascades as a novel therapeutic target for ischemic SCI and identify WIN55212-2 with the potential to treat ischemic SCI by targeting this pathway.

Affinity‐Driven Covalent Modulator of the Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) Cascade

AbstractTraditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)‐(+)‐isochaihulactone (1) exhibited significant inhibition against multiple‐drug‐resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off‐target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β‐unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI‐oriented activity‐based proteome profiling. By MS/MS and computer‐guided molecular biology approaches, an affinity‐driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the “ON/OFF” switch of apoptosis through non‐canonically nuclear GAPDH translocation, which bypasses the common apoptosis‐resistant route of MDR cancers.

PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme’s active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer’s disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals during cellular stress via several post-translational modifications that change its folding properties, protein-protein interactions and sub-cellular localization. We examined GAPDH properties in acute mouse liver injury due to ethanol and/or acetaminophen (APAP) treatment. Synergistic robust and time-dependent nuclear accumulation and aggregation of GAPDH were observed only in combined, but not individual, ethanol/APAP treatments. The small molecule GAPDH-targeting compound TCH346 partially attenuated liver damage possibly via mitochondrial mechanisms, and independent of nuclear accumulation and aggregation of GAPDH. These findings provide a novel potential mechanism for hepatotoxicity caused by combined alcohol and acetaminophen exposure.

GAPDH/Siah1 cascade is involved in traumatic spinal cord injury and could be attenuated by sivelestat sodium

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/Siah1 signaling pathway has been recognized as a sensor of nitric oxide (NO). It is associated with a variety of injurious conditions, suggesting its therapeutic potential for spinal cord injury (SCI). Sivelestat sodium (SIV), a neutrophil elastase (NE) inhibitor initially used to treat acute lung injury, has been known to protect against compression-induced and ischemic SCI. However, little is known about the relationship between the GAPDH/Siah1 cascade and SIV. Thus, we aimed to assess the role of GAPDH/Siah1 cascade in traumatic SCI and its possible link with SIV. Rats were assigned to four groups: sham group, SCI group, 5-mg/kg SIV group, and 10-mg/kg SIV. The traumatic SCI was induced by dropping a 10-g impactor from a height of 25mm on the dorsal surface of T9 and T10. SIV was injected intraperitoneally immediately after surgery. Our results showed that the nuclear translocation of GAPDH was induced together with the nuclear translocation of Siah1 and the formation of the GAPDH/Siah1 complex in the spinal cord after traumatic SCI. However, the activation of the GAPDH/Siah1 cascade was attenuated by treatment with SIV. We also found that SIV suppressed apoptosis, NE and inducible nitric oxide synthase (iNOS) protein expressions, the number of NE and iNOS immunostained cells, the production of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), and the activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) signaling in the spinal cord. The behavioral tests showed that SIV promoted functional recovery after traumatic SCI as reflected in the sustained increase in the Basso–Beattie–Bresnahan (BBB) scores throughout the observation period. In conclusion, our results reveal GAPDH/Siah1 as a novel signaling pathway during the progression of SCI, which can be blocked by SIV.

Acetaminophen (APAP) hepatocyte toxicity is associated with increased translocation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to the nucleus.

Previously, we have described that APAP intoxication induces nuclear translocation of GAPDH in rat liver. Recently, GAPDH nuclear traslocation was highlighted as a mediator of cell death.AimTo test if GAPDH nuclear translocation is associated with loss of viability of rat hepatocytes exposed to APAP. M&M: Rat isolated hepatocytes were exposed to different doses of APAP (0‐50 mM), for 24 h, in the absence or presence of RAS (10 µM), an inhibitor of GAPDH nuclear translocation. Cell viability was evaluated by the MTT assay and IC50 was calculated. Nuclear and cytosolic GAPDH, cytosolic cytochrome c, and mitochondrial BAX contents were evaluated by WB in hepatocytes treated with APAP (30 mM) and/or RAS (10 µM) for 24 h.ResultsIC50: RAS: 40.0±8.5 mM*; Control: 18.5±0.6 mM. Nuclear/Nuclear+cytosolic GAPDH expression ratio: APAP: 155±29*$; APAP‐RAS: 26±10*; RAS: 42±17*; C: 100±5. Cytosolic cytochrome c: APAP: 320±7.5*$; APAP‐RAS: 31±22; RAS: 19±32; C: 100±59. Mitochondrial BAX: APAP: 468±57*♦; APAP‐RAS: 342±45*♦; RAS: 152±81; C: 100±59. WB data are expressed as % of control. * P<0.05 vs C; $ P<0.05 vs RAS and APAP‐RAS. ♦ P<0.05 vs RAS.ConclusionAPAP acute intoxication increased GAPDH nuclear translocation in isolated hepatocytes. RAS blockage of such traslocation was associated with prevention in alteration of apoptotic/necroptotic marker (cytochrome C) and consequently with increased survival of the cells to APAP cytotoxicity. The data strongly suggest a link between APAP liver toxicity and GAPDH translocation.

Poly-ADP-Ribose-Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes

Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes and ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). db/db mice underwent 1.5 hours of hind limb ischemia followed by 1, 7, or 24 hours of reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24-hour period; the untreated group received Lactated Ringer (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity/intracellular localization, and poly-ADP-ribosylation of GAPDH. PARP activity was significantly lower in the PJ34-treated groups than in the Lactated Ringer group at 7 and 24 hours of reperfusion. There was significantly less muscle fiber injury in the PJ34-treated group than in the Lactated Ringer-treated mice at 24 hours of reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7 hours and 24 hours of IR. There were significant increases in metabolic activity only at 24 hours of IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly-ADP-ribosylation, and nuclear translocation of GAPDH. PJ34 reduced PARP activity, GAPDH ribosylation, and GAPDH translocation; ameliorated muscle fiber injury; and increased metabolic activity after hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy after IR in diabetic humans.

Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.

Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos

Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress–induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson’s disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations.