Aggregation Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Combination of a Chaperone Synthesis Inducer and an Inhibitor of GAPDH Aggregation for Rehabilitation after Traumatic Brain Injury: A Pilot Study

The recovery period after traumatic brain injury (TBI) is often complicated by secondary damage that may last for days or even months after trauma. Two proteins, Hsp70 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were recently described as modulating post-traumatic processes, and in this study, we test them as targets for combination therapy using an inhibitor of GAPDH aggregation (derivative of hydrocortisone RX624) and an inducer of Hsp70 synthesis (the pyrrolylazine derivative PQ-29). The protective effect of the combination on C6 rat glioblastoma cells treated with the cerebrospinal fluid of traumatized animals resulted in an increase in the cell index and in a reduced level of apoptosis. Using a rat weight drop model of TBI, we found that the combined use of both drugs prevented memory impairment and motor deficits, as well as a reduction of neurons and accumulation of GAPDH aggregates in brain tissue. In conclusion, we developed and tested a new approach to the treatment of TBI based on influencing distinct molecular mechanisms in brain cells.

MsrB1-regulated GAPDH oxidation plays programmatic roles in shaping metabolic and inflammatory signatures during macrophage activation.

Classically activated pro-inflammatory macrophages are generated from naive macrophages by pro-inflammatory cues that dynamically reprogram their fuel metabolism toward glycolysis. This increases their intracellular reactive oxygen species (ROS) levels, which then activate the transcription and release of pro-inflammatory mediators. Our study on mice that lack methionine sulfoxide reductase (Msr)-B1 shows that the resulting partial loss of protein methionine reduction in pro-inflammatory macrophages creates a unique metabolic signature characterized by altered fuel utilization, including glucose and pyruvate. This change also associates with hyper-inflammation that is at least partly due to sustained oxidation of an exposed methionine residue (M44) on glyceraldehyde 3-phosphate dehydrogenase (GAPDH), thereby inducing GAPDH aggregation, inflammasome activation, and subsequent increased interleukin (IL)-1β secretion. Since MsrB1-knockout mice exhibit increased susceptibility to lipopolysaccharide (LPS)-induced sepsis, the MsrB1-GAPDH axis may be a key molecular mechanism by which protein redox homeostasis controls the metabolic profile of macrophages and thereby regulates their functions.

In vitro stability of various enzymes by proline from H2O2 mediated oxidative damage

Plants under stress need to favour certain pathways so as to survive the stress period. Protection of specific enzymes by proline and other osmolytes could be one such mechanism to favour some pathways/processes. Therefore, the influence of osmolyte proline on conformational changes of various proteins caused by hydrogen peroxide (H2O2) was studied by intrinsic and extrinsic fluorescence emissions. H2O2 caused conformational change in proteins. Results indicated that for Alcohol dehydrogenase (AD) and Glutamate dehydrogenase (GD) enzymes, H2O2 induced conformational change was high and that for Glucose 6-phosphate dehydrogenase (G6PDH) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was low. Fluorescence and far-UV, CD measurements of catalase demonstrated that the H2O2 stabilized the protein secondary structure at low concentrations but destabilized it at higher concentrations. Intrinsic and ANS fluorescence results showed that proline at a concentration of 1.0 M prompted a reduction in the H2O2-induced exposed hydrophobic surfaces of studied enzymes, to different degrees which suggests its differential protective effect. Furthermore, SDS-PAGE studies revealed that proline was not able to reduce or inhibit the H2O2 mediated aggregation of GAPDH.

Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.

Regulation of Autophagy by Nuclear GAPDH and Its Aggregates in Cancer and Neurodegenerative Disorders.

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein–protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.

GAPDH-targeted therapy – A new approach for secondary damage after traumatic brain injury on rats

Massive neuronal death caused by a neurodegenerative pathology or damage due to ischaemia or traumatic brain injury leads to the appearance of cytosolic proteins in the extracellular space. We found that one of the most abundant cellular polypeptides, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), appearing in the medium of dying cells or body fluids is able to form aggregates that are cytotoxic to adjacent cells. Since we previously showed that the hydrocortisone derivative RX624 can inhibit the ability of GAPDH to transport the enzyme complex with polyglutamine and reduce the cytotoxicity of the complex, we explored the effects of GAPDH on SH-SY5Y neuroblastoma cells. We found that the latter treated with particular forms of GAPDH molecules die with a high efficiency, suggesting that the exogenous enzyme does kill adjacent cells. RX624 prevented the interaction of exogenous GAPDH with the cell membrane and reduced the level of death by more than 10%. We also demonstrated the efficiency of RX624 treatment in a rat model of traumatic brain injury. The chemical blocked the formation of GAPDH aggregates in the brain, inhibited the cytotoxic effects of cerebrospinal fluid and rescued the motor function of injured rats. Importantly, RX624 treatment of rats had a similar effect as the intracranial injection of anti-GAPDH antibodies.

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.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde

BackgroundThe aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an endogenous neurotoxin implicated in Parkinson's Disease. Elucidating protein targets of DOPAL is essential in understanding it's pathology. The enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of DOPAL. MethodsGAPDH activity was measured via reduction of NAD+ cofactor (340 nm). Protein aggregation was assessed with SDS-PAGE methods and specific modification via chemical probes. ResultsLow micromolar levels of DOPAL caused extensive GAPDH aggregation and irreversibly inhibited enzyme activity. The inactivation of GAPDH was dependent on both the catechol and aldehyde moieties of DOPAL. It is suggested that Cys are modified and oxidized by DOPAL. ConclusionsThe mechanism by which DOPAL modifies GAPDH can serve as a mechanistic explanation to the pathological events in Parkinson's Disease.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.

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.

Glyceraldehyde-3-phosphate dehydrogenase: Aggregation mechanisms and impact on amyloid neurodegenerative diseases

The review analyses data on specific features of aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and possible role of this enzyme in the development of neurodegenerative diseases. Different post-translational modifications of the enzyme are considered: oxidation, nitrosylation, and S-glutathionylation of the active site sulfhydryl groups, as well as phosphorylation, glycation and homocysteinylation of other amino acid residues. Modification of the sulfhydryl groups of the enzyme inhibits the enzymatic activity of GAPDH, resulting in slowdown of glycolysis, and may lead to the dissociation of the cofactor NAD from the active site of the enzyme. The resulting apo-GAPDH (without NAD) is less stable and prone to dissociation, denaturation, and subsequent aggregation. These processes could play a crucial role in the translocation of GAPDH subunits from the cytoplasm into the nucleus, which is linked to the induction of apoptosis. Phosphorylation and glycation of GAPDH are presumably involved in the regulation of protein-protein interactions and intracellular localization of the enzyme. Besides, glycation by dicarbonyl compounds and aldehydes may directly inhibit glycolysis. Homocysteinylation of GAPDH may stabilize aggregates of the enzyme by additional disulfide bonding. All types of post-translational modifications affect aggregation of GAPDH. A special attention is given to the role of chaperones in the amyloidogenic transformation of proteins and to confirmation of the hypothesis on blocking of the chaperones by misfolded protein forms. The denatured GAPDH forms were shown to interact directly with amyloidogenic proteins (alpha-synuclein and amyloid-beta peptide) and to play a crucial role in blocking of chaperone system.

Radicalization of Glyceraldehyde-3-Phosphate Dehydrogenase by HOCl in Living Cells

A number of post-translational oxidative modifications of the enzyme “cell-redox sensor” glyceraldehyde-3- phosphate dehydrogenase (GAPDH) have been reported. These modifications affect GAPDH structure, function, and cell fate; however no free-radical mechanisms have been reported in these processes. Herein we used the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-based spin trapping techniques to examine a novel free radical mechanism that causes GAPDH inactivation and aggregation in RAW264.7 cells primed with lipopolysaccharide (LPS). In these primed cells, GAPDH is oxidized by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) resulting in loss of enzyme activity and aggregation, accumulation of lactate and cell death. Due to the close spatial and physical proximity between MPO and GAPDH, and the oxidizing potential of HOCl, it may be the main species that triggers radicalization of GAPDH that ultimately results in enzyme aggregation and inactivation in LPS-primed macrophages. Lysine residues are the primary radicalization sites formed upon reaction of HOCl with the enzyme. Our data highlight the important relationship between radicalization of GAPDH and fate of stressed cells, which might help teasing out the cell response to stress at sites of inflammation.

Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most abundant targets of the oxidative stress. Oxidation of the enzyme causes its inactivation and the formation of intermolecular disulfide bonds, and leads to the accumulation of GAPDH aggregates and ultimately to cell death. The aim of this work was to reveal the ability of chemicals to break the described above pathologic linkage by inhibiting GAPDH aggregation. Using the model of oxidative stress based on SK-N-SH human neuroblastoma cells treated with hydrogen peroxide, we found that lentivirus-mediated down- or up-regulation of GAPDH content caused inhibition or enhancement of the protein aggregation and respectively reduced or increased the level of cell death. To reveal substances that are able to inhibit GAPDH aggregation, we developed a special assay based on dot ultrafiltration using the collection of small molecules of plant origin. In the first round of screening, five compounds were found to possess anti-aggregation activity as established by ultrafiltration and dynamic light scattering; some of the substances efficiently inhibited GAPDH aggregation in nanomolar concentrations. The ability of the compounds to bind GAPDH molecules was proved by the drug affinity responsive target stability assay, molecular docking and differential scanning calorimetry. Results of experiments with SK-N-SH human neuroblastoma treated with hydrogen peroxide show that two substances, RX409 and RX426, lowered the degree of GAPDH aggregation and reduced cell death by 30%. Oxidative injury was emulated in vivo by injecting of malonic acid into the rat brain, and we showed that the treatment with RX409 or RX426 inhibited GAPDH-mediated aggregation in the brain, reduced areas of the injury as proved by magnetic resonance imaging, and augmented the behavioral status of the rats as established by the “beam walking” test. In conclusion, the data show that two GAPDH binders could be therapeutically relevant in the treatment of injuries stemming from hard oxidative stress.

Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death.

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.

Kinetics of aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle. Effect of agents possessing chaperone-like activity

An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-β-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-β-CD reveals a purely protective effect.

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.

Effect of 2-hydroxypropyl-β-cyclodextrin on thermal inactivation, denaturation and aggregation of glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle

The effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on thermal aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle at 45 °C has been studied using dynamic light scattering. In the presence of HP-β-CD higher values of the rate of aggregation and larger aggregates were registered. The acceleration of GAPDH aggregation was due to destabilization of the enzyme molecule under the action of HP-β-CD. This is evidenced by the data on thermal inactivation of GAPDH and differential scanning calorimetry.

Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-induced Cell Death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(2) is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC(50) = 202 microM). Low concentrations of dopamine (50-100 microM) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200-300 microM)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 microM). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.