G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. METHODS: An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. RESULTS: The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. CONCLUSION: Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation.

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.

Glucose-6-Phosphatase-Dehydrogenase activity as modulative association between Parkinson's disease and periodontitis.

The association between periodontitis (PD) and Parkinson's disease (PK) is discussed due to the inflammatory component of neurodegenerative processes. PK severity and affected areas were determined using the following neuropsychological tests: Unified Parkinson's Disease Rating Score (UPDRS) and Hoehn and Yahr; non-motoric symptoms by Non-Motor Symptoms Scale (NMSS), and cognitive involvement by Mini-Mental State Examination (MMSE). Neuroinflammation and the resulting Glucose-6-Phosphatase-Dehydrogenase (G6PD) dysfunction are part of the pathophysiology of PK. This study aimed to evaluate these associations in periodontal inflammation. Clinical data and saliva-, serum-, and RNA-biobank samples of 50 well-characterized diametric patients with PK and five age- and sex-matched neurologically healthy participants were analyzed for G6PD function, periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, and Filifactor alocis), monocyte chemoattractant protein (MCP) 1, and interleukin (IL) 1-beta. Regression analysis was used to identify associations between clinical and behavioral data, and t-tests were used to compare health and disease. Compared with PK, no pathogens and lower inflammatory markers (p < 0.001) were detectible in healthy saliva and serum, PK-severity/UPDRS interrelated with the occurrence of Prevotella intermedia in serum as well as IL1-beta levels in serum and saliva (p = 0.006, 0.019, 0.034), Hoehn and Yahr correlated with Porphyromonas gingivalis, Prevotella intermedia, RNA IL1-beta regulation, serum, and saliva IL1-beta levels, with p-values of 0.038, 0.011, 0.008, <0.001, and 0.010, while MMSE was associated with Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, serum MCP 1 levels, RNA IL1-beta regulation and G6PD serum activity (p = 0.036, 0.003, 0.045, <0.001, and 0.021). Cognitive and motor skills seem to be important as representative tests are associated with periodontal pathogens and oral/general inflammation, wherein G6PD-saliva dysfunction might be involved. Clinical trial registration: https://www.bfarm.de/DE/Das-BfArM/Aufgaben/Deutsches-Register-Klinischer-Studien/_node.html, identifier DRKS00005388.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

BACKGROUND: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices.

The pentose phosphate pathway (PPP) is one of three major pathways involved in glucose metabolism, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) controls NADPH formation. NADPH, in turn, regulates the balance of oxidative stress and reactive oxygen species (ROS) levels. G6PD dysfunction, affecting the PPP, is implicated in neurological disorders, including epilepsy. However, PPP's role in epileptogenesis and ROS production during epileptic activity remains unclear. To clarify these points, we conducted electrophysiological and imaging analyses on mouse hippocampal brain slices. Using the specific G6PD inhibitor G6PDi-1, we assessed its effects on mouse hippocampal slices, examining intracellular ROS, glucose/oxygen consumption, the NAD(P)H level and ROS production during synaptic stimulation and in the 4AP epilepsy model. G6PDi-1 increased basal intracellular ROS levels and reduced synaptically induced glucose consumption but had no impact on baselevel of NAD(P)H and ROS production from synaptic stimulation. In the 4AP model, G6PDi-1 did not significantly alter spontaneous seizure frequency or H2O2 release amplitude but increased the frequency and peak amplitude of interictal events. These findings suggest that short-term PPP inhibition has a minimal impact on synaptic circuit activity.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Substitution of arginine 219 by glycine compromises stability, dimerization, and catalytic activity in a G6PD mutant.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies in humans, present in approximately half a billion people worldwide. More than 230 clinically relevant G6PD mutations of different classes have been reported to date. We hereby describe a patient with chronic hemolysis who presents a substitution of arginine by glycine at position 219 in G6PD protein. The variant was never described in an original publication or characterized on a molecular level. In the present study, we provide structural and biochemical evidence for the molecular basis of its pathogenicity. When compared to the wild-type enzyme, the Arg219Gly mutation markedly reduces the catalytic activity by 50-fold while having a negligible effect on substrate binding affinity. The mutation preserves secondary protein structure, but greatly decreases stability at higher temperatures and to trypsin digestion. Size exclusion chromatography elution profiles show monomeric and dimeric forms for the mutant, but only the latter for the wild-type form, suggesting a critical role of arginine 219 in G6PD dimer formation. Our findings have implications in the development of small molecule activators, with the goal of rescuing the phenotype observed in this and possibly other related mutants.

The Emerging Roles of the Metabolic Regulator G6PD in Human Cancers.

Metabolic reprogramming, especially reprogrammed glucose metabolism, is a well-known cancer hallmark related to various characteristics of tumor cells, including proliferation, survival, metastasis, and drug resistance. Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway (PPP), a branch of glycolysis, that converts glucose-6-phosphate (G6P) into 6-phosphogluconolactone (6PGL). Furthermore, PPP produces ribose-5-phosphate (R5P), which provides sugar-phosphate backbones for nucleotide synthesis as well as nicotinamide adenine dinucleotide phosphate (NADPH), an important cellular reductant. Several studies have shown enhanced G6PD expression and PPP flux in various tumor cells, as well as their correlation with tumor progression through cancer hallmark regulation, especially reprogramming cellular metabolism, sustaining proliferative signaling, resisting cell death, and activating invasion and metastasis. Inhibiting G6PD could suppress tumor cell proliferation, promote cell death, reverse chemoresistance, and inhibit metastasis, suggesting the potential of G6PD as a target for anti-tumor therapeutic strategies. Indeed, while challenges-including side effects-still remain, small-molecule G6PD inhibitors showing potential anti-tumor effect either when used alone or in combination with other anti-tumor drugs have been developed. This review provides an overview of the structural significance of G6PD, its role in and regulation of tumor development and progression, and the strategies explored in relation to G6PD-targeted therapy.

Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

PBX3 promotes pentose phosphate pathway and colorectal cancer progression by enhancing G6PD expression.

Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted G6PD transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential in vitro and in vivo. Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.

Musashi-2 (MSI2) promotes neuroblastoma tumorigenesis through targeting MYC-mediated glucose-6-phosphate dehydrogenase (G6PD) transcriptional activation.

Neuroblastoma (NB) is the deadliest pediatric solid tumor due to its rapid proliferation. Aberrant expression of MYCN is deemed as the most remarkable feature for the predictive hallmark of NB progression and recurrence. However, the phenomenon that only detection of MYCN in the nearly 20% of NB patients hints that there should be other vital oncogenes in the progression of NB. Here, we firstly show that MSI2 mRNA is augmented by analyzing public GEO datasets in the malignant stage according to International Neuroblastoma Staging System (INSS) stages. Although accumulating evidences uncover the emerging roles of MSI2 in several cancers, the regulatory functions and underlying mechanisms of MSI2 in NB remain under-investigated. Herein, we identified that high-expressed MSI2 and low-expressed n-Myc group account for 43.1% of total NB clinical samples (n = 65). Meanwhile, MSI2 expression is profoundly upregulated along with NB malignancy and negatively associated with the survival outcome of NB patients in the NB tissue microarray (NB: n = 65; Ganglioneuroblastoma: n = 31; Ganglioneuroma: n = 27). In vitro, our results revealed that MSI2 promoted migration, invasion, and proliferation of NB cells via enhancing pentose phosphate pathway. Mechanistically, MSI2 upregulated the key enzyme glucose-6-phosphate dehydrogenase (G6PD) via directly binding to 3'-untranslated regions of c-Myc mRNA to facilitate its stability, resulting in enhancing pentose phosphate pathway. Our findings reveal that MSI2 promotes pentose phosphate pathway via activating c-Myc-G6PD signaling, suggesting that MSI2 exhibits a novel and powerful target for the diagnosis and treatment of NB.

Glucose-6-phosphate dehydrogenase: a therapeutic target for ovarian cancer.

INTRODUCTION: Ovarian cancer (OC) is a gynecological tumor disease, which is usually diagnosed at an advanced stage and has a poor prognosis. It has been established that the glucose metabolism rate of cancer cells is significantly higher than that of normal cells, and the pentose phosphate pathway (PPP) is an important branch pathway for glucose metabolism. Glucose-6-phosphate dehydrogenase (G6PD) is the key rate-limiting enzyme in the PPP, which plays an important role in the initiation and development of cancer (such as OC), and has been considered as a promisinganti-cancer target. AREAS COVERED: In this review, based on the structure and biological function of G6PD, recent research on the roles of G6PD in the progression, metastasis, and chemoresistance of OC are summarized and accompanied by proposed molecular mechanisms, which may provide a systematic understanding of targeting G6PD for the treatment of patients with OC. EXPERT OPINION: Accumulating evidence demonstrates that G6PD is a promising target of cancer. The development of G6PD inhibitors for cancer treatment merits broad application prospects.

G6PDi-1 is a Potent Inhibitor of G6PDH and of Pentose Phosphate pathway-dependent Metabolic Processes in Cultured Primary Astrocytes.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyses the rate limiting first step of the oxidative part of the pentose phosphate pathway (PPP), which has a crucial function in providing NADPH for antioxidative defence and reductive biosyntheses. To explore the potential of the new G6PDH inhibitor G6PDi-1 to affect astrocytic metabolism, we investigated the consequences of an application of G6PDi-1 to cultured primary rat astrocytes. G6PDi-1 efficiently inhibited G6PDH activity in lysates of astrocyte cultures. Half-maximal inhibition was observed for 100 nM G6PDi-1, while presence of almost 10 µM of the frequently used G6PDH inhibitor dehydroepiandrosterone was needed to inhibit G6PDH in cell lysates by 50%. Application of G6PDi-1 in concentrations of up to 100 µM to astrocytes in culture for up to 6 h did not affect cell viability nor cellular glucose consumption, lactate production, basal glutathione (GSH) export or the high basal cellular ratio of GSH to glutathione disulfide (GSSG). In contrast, G6PDi-1 drastically affected astrocytic pathways that depend on the PPP-mediated supply of NADPH, such as the NAD(P)H quinone oxidoreductase (NQO1)-mediated WST1 reduction and the glutathione reductase-mediated regeneration of GSH from GSSG. These metabolic pathways were lowered by G6PDi-1 in a concentration-dependent manner in viable astrocytes with half-maximal effects observed for concentrations between 3 and 6 µM. The data presented demonstrate that G6PDi-1 efficiently inhibits the activity of astrocytic G6PDH and impairs specifically those metabolic processes that depend on the PPP-mediated regeneration of NADPH in cultured astrocytes.

A GSTP1-mediated lactic acid signaling promotes tumorigenesis through the PPP oxidative branch.

Lactic acidosis is a feature of solid tumors and plays fundamental role(s) rendering cancer cells to adapt to diverse metabolic stresses, but the mechanism underlying its roles in redox homeostasis remains elusive. Here we show that G6PD is phosphorylated at tyrosine 249/322 by the SRC through the formation of a GSTP1-G6PD-SRC complex. Lactic acid attenuates this formation and the phosphorylation of G6PD by non-covalently binding with GSTP1. Furthermore, lactic acid increases the activity of G6PD and facilitates the PPP (NADPH production) through its sensor GSTP1, thereby exhibiting resistance to reactive oxygen species when glucose is scarce. Abrogating a GSTP1-mediated lactic acid signaling showed attenuated tumor growth and reduced resistance to ROS in breast cancer cells. Importantly, positive correlations between immuno-enriched SRC protein and G6PD Y249/322 phosphorylation specifically manifest in ER/PR positive or HER negative types of breast cancer. Taken together, these results suggest that GSTP1 plays a key role in tumor development by functioning as a novel lactate sensor.

Effects of salt stress on seed germination and respiratory metabolism in different Flueggea suffruticosa genotypes.

The selection and utilization of ornamental plants that are highly tolerant to salt are helpful for landscape construction and the ecological protection of coastal and arid areas. To evaluate salt tolerance, one of the most used methods is the observation of seed germination under salt stress. Therefore, this work aimed to evaluate the influence of different concentrations of NaCl in water absorption, germination, and respiratory metabolism in seeds of different Flueggea suffruticosa genotypes. P2 and P27, salt-sensitive and salt-tolerant line s of F. suffruticosa, were chosen for treatment with 0, 40, 80, 120, 160, 200, and 240 mM NaCl. F. suffruticosa under salt stress exhibited inhibition of seed germination. The seeds of F. suffruticosa have different times for the physiological phases of water absorption with different NaCl concentrations. Salt stress retarded the seed water absorption process, and it depended on seed genotypes for F. suffruticosa. Soluble sugars accumulated in both P2 and P27 under salt stress. Meanwhile, the activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase, citrate synthase, and glucose-6-phosphate dehydrogenase were overall increased in P27 after salt treatment, which caused increases in pyruvic acid and citric acid. The citrate synthase and glucose-6-phosphate dehydrogenase activities decreased in P2. These results suggest that the respiratory metabolism of salt-tolerant F. suffruticosa was enhanced, compared with the salt-sensitive line, to ameliorate the repression of seed germination under salt stress. The different changes in respiratory metabolism could influence the degree of salt tolerance.

Rapid phosphorylation of glucose-6-phosphate dehydrogenase by casein kinase 2 sustains redox homeostasis under ionizing radiation.

Exposure to ionizing radiation leads to oxidative damages in living cells. NADPH provides the indispensable reducing power to regenerate the reduced glutathione to maintain cellular redox equilibria. In mammalian cells, pentose phosphate pathway (PPP) is the major route to produce NADPH by using glycolytic intermediates, and the rate-limiting step of PPP is controlled by glucose-6-phosphate dehydrogenase (G6PD). Nevertheless, whether G6PD is timely co-opted under ionizing radiation to cope with oxidative stress remains elusive. Here we show that cellular G6PD activity is induced 30 min after ionizing radiation, while its protein expression is mostly unchanged. Mechanistically, casein kinase 2 (CK2) phosphorylates G6PD T145 under ionizing radiation, which consolidates the enzymatic activity of G6PD by facilitating G6PD binding with its substrate NADP+. Further, CK2-dependent G6PD T145 phosphorylation promotes NADPH production, decreases ROS level and supports cell proliferation under ionizing radiation. Our findings report a new anti-oxidative signaling route under ionizing radiation, by which CK2-mediated rapid activation of G6PD orchestrates NADPH synthesis to maintain redox homeostasis, thereby highlighting its potential value in the early treatment of ionizing radiation-induced injuries.

Reductive stress induced by NRF2/G6PD through glucose metabolic reprogramming promotes malignant transformation in Arsenite-exposed human keratinocytes.

Our previous research found that the nuclear factor-E2-related factor 2 (NRF2) protein was sustained activated in malignant transformation of human keratinocyte (HaCaT cells) caused by NaAsO2, but the role of NRF2 in it remains unknown. In this study, malignant transformation of HaCaT cells and labeled HaCaT cells used to detect mitochondrial glutathione levels (Mito-Grx1-roGFP2 HaCaT cells) were induced by 1.0 µM NaAsO2. Redox levels were measured at passages 0, early stage (passages 1, 7, 14), later stage (passages 21, 28 and 35) of arsenite-treated HaCaT cells. Oxidative stress levels increased at early stage. The NRF2 pathway was sustained activated. Cells and mitochondrial reductive stress levels (GSH/GSSG and NADPH/NADP+) increased. The mitochondrial GSH/GSSG levels of Mito-Grx1-roGFP2 HaCaT cells also increased. The indicators of glucose metabolism glucose-6-phosphate, lactate and the glucose-6-phosphate dehydrogenase (G6PD) levels increased, however Acetyl-CoA level decreased. Expression levels of glucose metabolic enzymes increased. After transfection with NRF2 siRNA, the indicators of glucose metabolism were reversed. After transfection with NRF2 or G6PD siRNA, cells and mitochondrial reductive stress levels decreased and the malignant phenotype was reversed. In conclusion, oxidative stress occurred in the early stage and the NRF2 was sustained high expression. In the later stage, increased NRF2/G6PD through glucose metabolic reprogramming induced reductive stress, thereby leading to malignant transformation.

A computational study of structural analysis of Class I human glucose-6-phosphate dehydrogenase (G6PD) variants: Elaborating the correlation to chronic non-spherocytic hemolytic anemia (CNSHA).

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect that affects more than 500 million people worldwide. Individuals affected with G6PD deficiency may occasionally suffer mild-to-severe chronic hemolytic anemia. Chronic non-spherocytic hemolytic anemia (CNSHA) is a potential result of the Class I G6PD variants. This comparative computational study attempted to correct the defect in variants structure by docking the AG1 molecule to selected Class I G6PD variants [G6PDNashville (Arg393His), G6PDAlhambra (Val394Leu), and G6PDDurham (Lys238Arg)] at the dimer interface and structural NADP+ binding site. It was followed by an analysis of the enzyme conformations before and after binding to the AG1 molecule using the molecular dynamics simulation (MDS) approach, while the severity of CNSHA was determined via root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bonds, salt bridges, radius of gyration (Rg), solvent accessible surface area analysis (SASA), and principal component analysis (PCA). The results revealed that G6PDNashville (Arg393His) and G6PDDurham (Lys238Arg) had lost the direct contact with structural NADP+ and salt bridges at Glu419 - Arg427 and Glu206 - Lys407 were disrupted in all selected variants. Furthermore, the AG1 molecule re-stabilized the enzyme structure by restoring the missing interactions. Bioinformatics approaches were also used to conduct a detailed structural analysis of the G6PD enzyme at a molecular level to understand the implications of these variants toward enzyme function. Our findings suggest that despite the lack of treatment for G6PDD to date, AG1 remains a novel molecule that promotes activation in a variety of G6PD variants.

Structure of Leishmania donovani 6-Phosphogluconate Dehydrogenase and Inhibition by Phosphine Gold(I) Complexes: A Potential Approach to Leishmaniasis Treatment.

As unicellular parasites are highly dependent on NADPH as a source for reducing equivalents, the main NADPH-producing enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway are considered promising antitrypanosomatid drug targets. Here we present the biochemical characterization and crystal structure of Leishmania donovani 6PGD (Ld6PGD) in complex with NADP(H). Most interestingly, a previously unknown conformation of NADPH is visible in this structure. In addition, we identified auranofin and other gold(I)-containing compounds as efficient Ld6PGD inhibitors, although it has so far been assumed that trypanothione reductase is the sole target of auranofin in Kinetoplastida. Interestingly, 6PGD from Plasmodium falciparum is also inhibited at lower micromolar concentrations, whereas human 6PGD is not. Mode-of-inhibition studies indicate that auranofin competes with 6PG for its binding site followed by a rapid irreversible inhibition. By analogy with other enzymes, this suggests that the gold moiety is responsible for the observed inhibition. Taken together, we identified gold(I)-containing compounds as an interesting class of inhibitors against 6PGDs from Leishmania and possibly from other protozoan parasites. Together with the three-dimensional crystal structure, this provides a valid basis for further drug discovery approaches.

G6PD activation in TNBC cells induces macrophage recruitment and M2 polarization to promote tumor progression.

Glucose-6-phosphate dehydrogenase (G6PD) is involved in triple-negative breast cancer (TNBC) progression. Metabolic crosstalk between cancer cells and tumor-associated macrophages mediates tumor progression in TNBC. Molecular biological methods were applied to clarify the mechanism of the crosstalk between TNBC cells and M2 macrophages. In the present study, we verified that G6PD overexpression drives M2 macrophage polarization by directly combining with phospho-STAT1 and upregulating CCL2 and TGF-ß1 secretion in TNBC cells. In turn, M2-like TAMs activated TNBC cells through IL-10 secretion, providing feedback to upregulate G6PD and promote TNBC cell migration and proliferation in vitro. Furthermore, we found that 6-AN (a specific inhibitor of G6PD) not only suppressed the cancer-driven polarization of macrophages toward the M2 phenotype but also inhibited the inherent M2 polarization of macrophages. Targeting the G6PD-regulated pentose phosphate pathway restrained TNBC progression and M2-type polarization of macrophages in vitro and in vivo.