GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

The role of GAPDH in the selective toxicity of CNP in melanoma cells.

BACKGROUND: Malignant melanoma is the most aggressive form of skin cancer with a rather poor prognosis. Standard chemotherapy often results in severe side effects on normal (healthy) cells finally being difficult to tolerate for the patients. Shown by us earlier, cerium oxide nanoparticles (CNP, nanoceria) selectively killed A375 melanoma cells while not being cytotoxic at identical concentrations on non-cancerous cells. In conclusion, the redox-active CNP exhibited both prooxidative as well as antioxidative properties. In that context, CNP induced mitochondrial dysfunction in the studied melanoma cells via generation of reactive oxygene species (primarily hydrogen peroxide (H2O2)), but that does not account for 100% of the toxicity. AIM: Cancer cells often show an increased glycolytic rate (Warburg effect), therefore we focused on CNP mediated changes of the glucose metabolism. RESULTS: It has been shown before that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity is regulated via oxidation of a cysteine in the active center of the enzyme with a subsequent loss of activity. Upon CNP treatment, formation of cellular lactate and GAPDH activity were significantly lowered. The treatment of melanoma cells and melanocytes with the GAPDH inhibitor heptelidic acid (HA) decreased viability to a much higher extent in the cancer cells than in the studied normal (healthy) cells, highlighting and supporting the important role of GAPDH in cancer cells. CONCLUSION: We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target protein for CNP mediated thiol oxidation.

Analyzing the interaction of Helicobacter pylori GAPDH with host molecules and hemin: Inhibition of hemin binding.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme. Apart from its primary role in the glycolytic pathway, in many bacterial species it is found in the extracellular milieu and also on the bacterial surface. Positioning on the bacterial surface allows the GAPDH molecule to interact with many host molecules such as plasminogen, fibrinogen, fibronectin, laminin and mucin etc. This facilitates the bacterial colonization of the host. Helicobacter pylori is a major human pathogen that causes a number of gastrointestinal infections and is the main cause of gastric cancer. The binding analysis of H. pylori GAPDH (HpGAPDH) with host molecules has not been carried out. Hence, we studied the interaction of HpGAPDH with holo-transferrin, lactoferrin, haemoglobin, fibrinogen, fibronectin, catalase, plasminogen and mucin using biolayer interferometry. Highest and lowest binding affinity was observed with lactoferrin (4.83 ± 0.70 × 10-9 M) and holo-transferrin (4.27 ± 2.39 × 10-5 M). Previous studies established GAPDH as a heme chaperone involved in intracellular heme trafficking and delivery to downstream target proteins. Therefore, to get insights into heme binding, the interaction between HpGAPDH and hemin was analyzed. Hemin binds to HpGAPDH with an affinity of 2.10 µM while the hemin bound HpGAPDH does not exhibit activity. This suggests that hemin most likely binds at the active site of HpGAPDH, prohibiting substrate binding. Blind docking of hemin with HpGAPDH also supports positioning of hemin at the active site. Metal ions were found to inhibit the activity of HpGAPDH, suggesting that it also possibly occupies the substrate binding site. Furthermore, with metal-bound HpGAPDH, hemin binding was not observed, suggesting metal ions act as an inhibitor of hemin binding. Since GAPDH has been identified as a heme chaperone, it will be interesting to analyse the biological consequences of inhibition of heme binding to GAPDH by metal ions.

Piperlongumine based nanomedicine impairs glycolytic metabolism in triple negative breast cancer stem cells through modulation of GAPDH & FBP1.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and exhibits high rate of chemoresistance, metastasis, and relapse. This can be attributed to the failure of conventional therapeutics to target a sub-population of slow cycling or quiescent cells called as cancer stem cells (CSCs). Therefore, elimination of CSCs is essential for effective TNBC treatment. PURPOSE: Research suggests that breast CSCs exhibit elevated glycolytic metabolism which directly contributes in maintenance of stemness, self-renewability and chemoresistance as well as in tumor progression. Therefore, this study aimed to target rewired metabolism which can serve as Achilles heel for CSCs population and have far reaching effect in TNBC treatment. METHODS: We used two preclinical models, zebrafish and nude mice to evaluate the fate of nanoparticles as well as the therapeutic efficacy of both piperlongumine (PL) and its nanomedicine (PL-NPs). RESULTS: In this context, we explored a phytochemical piperlongumine (PL) which has potent anti-cancer properties but poor pharmacokinetics impedes its clinical translation. So, we developed PLGA based nanomedicine for PL (PL-NPs), and demonstrated that it overcomes the pharmacokinetic limitations of PL, along with imparting advantages of selective tumor targeting through Enhanced Permeability and Retention (EPR) effect in zebrafish xenograft model. Further, we demonstrated that PL-NPs efficiently inhibit glycolysis in CSCs through inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by modulating glutathione S-transferase pi 1 (GSTP1) and upregulation of fructose-1,6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis. We also illustrated that inhibition of glycolysis results in overall tumor regression in two preclinical models. CONCLUSION: This study discusses novel mechanism of action by which PL acts on CSCSs. Taken together our study provides insight into development of PL based nanomedicine which could be exploited in clinics to achieve complete eradication of TNBC by targeting CSCs.

Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.

Subcutaneous Streptococcus dysgalactiae GAPDH vaccine in mice induces a proficient innate immune response.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. OBJECTIVE: This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. METHODS: Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. RESULTS: Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. CONCLUSIONS: GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.

[Diffuse large B-cell lymphoma expressing high-level glyceraldehyde 3-phosphate dehydrogenase complicated with hypoglycemia].

A 69-year-old male patient was referred to our hospital for further examination of hypoglycemia, splenomegaly, and para-aortic lymphadenopathy. The patient was diagnosed with diffuse large B-cell lymphoma (DLBCL) by para-aortic lymph node biopsy. Hypoglycemia was refractory to glucose supplementation but improved shortly after chemotherapy. This situation suggested that hypoglycemia was caused by lymphoma. We compared the expression levels of glyceraldehyde 3-phosphate dehydrogenase, a glycolytic enzyme whose expression is positively correlated with the glycolytic activity of cells, between the current case and two cases of DLBCL without hypoglycemia to explore the possibility that hypoglycemia was due to intense glucose consumption by lymphoma cells through their high glycolytic activity. Results revealed substantially higher expression levels of glyceraldehyde 3-phosphate dehydrogenase in the current case than in DLBCL without hypoglycemia, suggesting that the glycolytic pathway was enhanced in the current case. These results implied that intense glucose consumption by lymphoma cells through their high glycolytic activity causes hypoglycemia.

Elevated FBXW10 drives hepatocellular carcinoma tumorigenesis via AR-VRK2 phosphorylation-dependent GAPDH ubiquitination in male transgenic mice.

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men, but the underlying mechanism remains to be further explored. Here, we report that ubiquitinated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is responsible for HCC tumorigenesis in males. Mechanistically, FBXW10 promotes GAPDH polyubiquitination and activation; VRK2-dependent phosphorylation of GAPDH Ser151 residue is critical for GAPDH ubiquitination and activation. Activated GAPDH interacts with TRAF2, leading to upregulation of the canonical and noncanonical NF-κB pathways, and increases PD-L1 and AR-VRK2 expression, followed by induction of immune evasion, HCC tumorigenesis, and metastasis. Notably, the GAPDH inhibitor koningic acid (KA) activates immune response and protects against FBXW10-driven HCC in vivo. In HCC clinical samples, the expression of active GAPDH is positively correlated with that of FBXW10 and VRK2. We propose that the FBXW10/AR/VRK2/GAPDH/NF-κB axis is critical for HCC tumorigenesis in males. Targeting this axis with KA is a potential therapeutic strategy for male HCC patients.

Measuring the Oxidation State and Enzymatic Activity of Glyceraldehyde Phosphate Dehydrogenase (GAPDH).

Glyceraldehyde phosphate dehydrogenase (GAPDH) is a highly conserved, essential, and abundant enzyme that catalyzes a rate-determining step of glycolysis. GAPDH catalyzes the nicotinamide adenine dinucleotide (NAD+)- and inorganic phosphate-dependent oxidation and phosphorylation of glyceraldehyde phosphate (GAP) to form 1,3-bisphosphoglycerate (BPG). As part of its mechanism of action, GAPDH employs a redox-sensitive cysteine that serves as a nucleophile to form a covalent adduct with GAP in order to set-up subsequent oxidation and phosphorylation steps. As a result of the redox sensitivity of the active site cysteine residue, GAPDH is susceptible to oxidative inactivation by oxidants such as hydrogen peroxide (H2O2). Indeed, numerous studies have demonstrated that oxidative inactivation of GAPDH has important metabolic consequences for adaptation to life in air and oxidative stress since decreased GAPDH activity results in the rerouting of carbon flux away from glycolysis and toward the pentose phosphate pathway to produce the key cellular reductant and antioxidant, NADPH. Thus, the ability to probe GAPDH oxidation and activity provides an important snapshot of the intracellular redox environment and glycolytic flux. Herein, we describe methods to measure reduced and oxidized GAPDH using thiol alkylation assays as well as GAPDH enzymatic activity.

The GAPDH redox switch safeguards reductive capacity and enables survival of stressed tumour cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response to hydrogen peroxide, leading to rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable to stimulate the oxidative pentose phosphate pathway and enhance their reductive capacity. Specifically, we find that anchorage-independent growth of cells and spheroids is limited by an elevation of endogenous peroxide levels and is largely dependent on a functional GAPDH redox switch. Likewise, tumour growth in vivo is limited by peroxide stress and suppressed when the GAPDH redox switch is disabled in tumour cells. The induction of additional intratumoural oxidative stress by chemo- or radiotherapy synergized with the deactivation of the GAPDH redox switch. Mice lacking the GAPDH redox switch exhibit altered fatty acid metabolism in kidney and heart, apparently in compensation for the lack of the redox switch. Together, our findings demonstrate the physiological and pathophysiological relevance of oxidative GAPDH inactivation in mammals.

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and ß cells from humans and mice.

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

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.

Origin of Elevated S-Glutathionylated GAPDH in Chronic Neurodegenerative Diseases.

H2O2-oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalytic cysteine residues (Cc(SH) undergo rapid S-glutathionylation. Restoration of the enzyme activity is accomplished by thiol/disulfide SN2 displacement (directly or enzymatically) forming glutathione disulfide (G(SS)G) and active enzyme, a process that should be facile as Cc(SH) reside on the subunit surface. As S-glutathionylated GAPDH accumulates following ischemic and/or oxidative stress, in vitro/silico approaches have been employed to address this paradox. Cc(SH) residues were selectively oxidized and S-glutathionylated. Kinetics of GAPDH dehydrogenase recovery demonstrated that glutathione is an ineffective reactivator of S-glutathionylated GAPDH compared to dithiothreitol. Molecular dynamic simulations (MDS) demonstrated strong binding interactions between local residues and S-glutathione. A second glutathione was accommodated for thiol/disulfide exchange forming a tightly bound glutathione disulfide G(SS)G. The proximal sulfur centers of G(SS)G and Cc(SH) remained within covalent bonding distance for thiol/disulfide exchange resonance. Both these factors predict inhibition of dissociation of G(SS)G, which was verified by biochemical analysis. MDS also revealed that both S-glutathionylation and bound G(SS)G significantly perturbed subunit secondary structure particularly within the S-loop, region which interacts with other cellular proteins and mediates NAD(P)+ binding specificity. Our data provides a molecular rationale for how oxidative stress elevates S-glutathionylated GAPDH in neurodegenerative diseases and implicates novel targets for therapeutic intervention.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Exploring the Role of Glycolytic Enzymes PFKFB3 and GAPDH in the Modulation of Aß and Neurodegeneration and Their Potential of Therapeutic Targets in Alzheimer's Disease.

Alzheimer's disease (AD) is presently the 6th major cause of mortality across the globe. However, it is expected to rise rapidly, following cancer and heart disease, as a leading cause of death among the elderly peoples. AD is largely characterized by metabolic changes linked to glucose metabolism and age-induced mitochondrial failure. Recent research suggests that the glycolytic pathway is required for a range of neuronal functions in the brain including synaptic transmission, energy production, and redox balance; however, alteration in glycolytic pathways may play a significant role in the development of AD. Moreover, it is hypothesized that targeting the key enzymes involved in glucose metabolism may help to prevent or reduce the risk of neurodegenerative disorders. One of the major pro-glycolytic enzyme is 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3); it is normally absent in neurons but abundant in astrocytes. Similarly, another key of glycolysis is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which catalyzes the conversion of aldolase and glyceraldehyde 3 phosphates to 1,3 bisphosphoglycerate. GAPDH has been reported to interact with various neurodegenerative disease-associated proteins, including the amyloid-ß protein precursor (AßPP). These findings indicate PFKFB3 and GAPDH as a promising therapeutic target to AD. Current review highlight the contributions of PFKFB3 and GAPDH in the modulation of Aßand AD pathogenesis and further explore the potential of PFKFB3 and GAPDH as therapeutic targets in AD.

Immunomodulatory effects of extracellular glyceraldehyde 3-phosphate dehydrogenase of exopolysaccharide-producing Lactiplantibacillus plantarum JCM 1149.

Probiotic lactic acid bacteria evoke immunomodulatory effects in the host; however, the reasons for the different effects of various species and strains remain to be elucidated. To clarify the critical immunomodulatory components and impact of exopolysaccharide (EPS) in Lactiplantibacillus plantarum, 11 types of L. plantarum strains were compared for the production of EPS, inflammatory cytokines, interleukin-6 and -12, and the anti-inflammatory cytokine, interleukin-10, from THP-1 differentiated dendritic cells. EPS in the fermented medium correlated with cytokine-inducing activities. L. plantarum JCM 1149, with the highest production of EPS, also induced interleukin-6, -10, and -12 among the 11 tested strains. Notably, the cytokine-producing activities overlapped with the protein fraction in gel filtration chromatography but not with EPS, which has been reported to exert immunomodulatory effects. The 41 kDa protein that coexisted with EPS was purified as a major active component and identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known moonlighting protein. GAPDH secretion was reduced when EPS synthesis inhibitors were added to the culture medium. RNA sequencing of GAPDH-treated THP-1 cells revealed an up-regulation in the expression of genes involved in transcriptional regulation, cell surface receptor signalling, immune response, and matrix components. Here, we report, to our knowledge for the first time, that the cell surface-associated L. plantarum GAPDH plays a crucial role in cytokine production in THP-1 cells, but EPS with less activity may help GAPDH secretion.

Modulations in human neutrophil metabolome and S-glutathionylation of glycolytic pathway enzymes during the course of extracellular trap formation.

Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, ß-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.

Unravelling the regulation pathway of photosynthetic AB-GAPDH.

Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC-SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein of Lactobacillus gasseri attenuates allergic asthma via immunometabolic change in macrophages.

BACKGROUND: The extra-intestinal effects of probiotics for preventing allergic diseases are well known. However, the probiotic components that interact with host target molecules and have a beneficial effect on allergic asthma remain unknown. Lactobacillus gasseri attenuates allergic airway inflammation through the activation of peroxisome proliferator- activated receptor γ (PPARγ) in dendritic cells. Therefore, we aimed to isolate and investigate the immunomodulatory effect of the PPARγ activation component from L. gasseri. METHODS: Culture supernatants of L. gasseri were fractionated and screened for the active component for allergic asthma. The isolated component was subjected to in vitro functional assays and then cloned. The crystal structure of this component protein was determined using X-ray crystallography. Intrarectal inoculation of the active component-overexpressing Clear coli (lipopolysaccharide-free Escherichia coli) and intraperitoneal injection of recombinant component protein were used in a house dust mite (HDM)-induced allergic asthma mouse model to investigate the protective effect. Recombinant mutant component proteins were assayed, and their structures were superimposed to identify the detailed mechanism of alleviating allergic inflammation. RESULTS: A moonlighting protein, glycolytic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LGp40, that has multifunctional effects was purified from cultured L. gasseri, and the crystal structure was determined. Both intrarectal inoculation of LGp40-overexpressing Clear coli and intraperitoneal administration of recombinant LGp40 protein attenuated allergic inflammation in a mouse model of allergic asthma. However, CDp40, GAPDH isolated from Clostridium difficile did not possess this anti-asthma effect. LGp40 redirected allergic M2 macrophages toward the M1 phenotype and impeded M2-prompted Th2 cell activation through glycolytic activity that induced immunometabolic changes. Recombinant mutant LGp40, without enzyme activity, showed no protective effect against HDM-induced airway inflammation. CONCLUSIONS: We found a novel mechanism of moonlighting LGp40 in the reversal of M2-prompted Th2 cell activation through glycolytic activity, which has an important immunoregulatory role in preventing allergic asthma. Our results provide a new strategy for probiotics application in alleviating allergic asthma.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.