RESUMO
Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Neoplasias do Colo/enzimologia , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/enzimologia , Melanoma Experimental/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Estabilidade Enzimática , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Microambiente Tumoral , Quinase Induzida por NF-kappaBRESUMO
B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.
Assuntos
Carbono/metabolismo , Glucose/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Estresse Oxidativo , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Via de Pentose Fosfato , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína Fosfatase 2/deficiência , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transcrição GênicaRESUMO
The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Gluconatos/metabolismo , Neoplasias/enzimologia , Proteína Fosfatase 2/metabolismo , Células A549 , Quinases Proteína-Quinases Ativadas por AMP , Animais , Proliferação de Células , Ativação Enzimática , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Células HEK293 , Células HT29 , Humanos , Células K562 , Células MCF-7 , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Via de Pentose Fosfato , Ligação Proteica , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribulosefosfatos/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Carga Tumoral , Quinases da Família src/metabolismoRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects over 500 million individuals who can experience anemia in response to oxidative stressors such as certain foods and drugs. Recently, the World Health Organization (WHO) called for revisiting G6PD variant classification as a priority to implement genetic medicine in low- and middle-income countries. Toward this goal, we sought to collect reports of G6PD variants and provide interpretations. We identified 1,341 G6PD variants in population and clinical databases. Using the ACMG standards and guidelines for the interpretation of sequence variants, we provided interpretations for 268 variants, including 186 variants that were not reported or of uncertain significance in ClinVar, bringing the total number of variants with non-conflicting interpretations to 400. For 414 variants with functional or clinical data, we analyzed associations between activity, stability, and current classification systems, including the new 2022 WHO classification. We corroborated known challenges with classification systems, including phenotypic variation, emphasizing the importance of comparing variant effects across individuals and studies. Biobank data made available by All of Us illustrate the benefit of large-scale sequencing and phenotyping by adding additional support connecting variants to G6PD-deficient anemia. By leveraging available data and interpretation guidelines, we created a repository for information on G6PD variants and nearly doubled the number of variants with clinical interpretations. These tools enable better interpretation of G6PD variants for the implementation of genetic medicine.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Saúde da População , Humanos , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Variação Biológica da População , CatalogaçãoRESUMO
Obesity is a major risk factor for liver and cardiovascular diseases. However, obesity-driven mechanisms that contribute to the pathogenesis of multiple organ diseases are still obscure and treatment is inadequate. We hypothesized that increased , glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme in the pentose shunt, is critical in evoking metabolic reprogramming in multiple organs and is a significant contributor to the pathogenesis of liver and cardiovascular diseases. G6PD is induced by a carbohydrate-rich diet and insulin. Long-term (8 months) high-fat diet (HFD) feeding increased body weight and elicited metabolic reprogramming in visceral fat, liver, and aorta, of the wild-type rats. In addition, HFD increased inflammatory chemokines in visceral fat. Interestingly, CRISPR-edited loss-of-function Mediterranean G6PD variant (G6PDS188F) rats, which mimic human polymorphism, moderated HFD-induced weight gain and metabolic reprogramming in visceral fat, liver, and aorta. The G6PDS188F variant prevented HFD-induced CCL7 and adipocyte hypertrophy. Furthermore, the G6PDS188F variant increased Magel2 - a gene encoding circadian clock-related protein that suppresses obesity associated with Prader-Willi syndrome - and reduced HFD-induced non-alcoholic fatty liver. Additionally, the G6PDS188F variant reduced aging-induced aortic stiffening. Our findings suggest G6PD is a regulator of HFD-induced obesity, adipocyte hypertrophy, and fatty liver.
Assuntos
Adipócitos , Dieta Hiperlipídica , Fígado Gorduroso , Glucosefosfato Desidrogenase , Hipertrofia , Obesidade , Animais , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Masculino , Ratos , Obesidade/metabolismo , Obesidade/genética , Obesidade/patologia , Obesidade/etiologia , Dieta Hiperlipídica/efeitos adversos , Adipócitos/metabolismo , Adipócitos/patologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/metabolismo , Fígado/patologia , Ratos Sprague-Dawley , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both in vitro and in vivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.
Assuntos
Carcinoma Ductal Pancreático , Desoxicitidina , Gencitabina , Glucosefosfato Desidrogenase , MicroRNAs , Neoplasias Pancreáticas , Receptores da Prolactina , Animais , Feminino , Humanos , Camundongos , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores da Prolactina/metabolismo , Receptores da Prolactina/genética , Camundongos NusRESUMO
Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.
Assuntos
Indicadores e Reagentes/análise , Microfluídica/métodos , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Testes Diagnósticos de Rotina , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Fluorometria , Glucosefosfato Desidrogenase/análise , Glucosefosfato Desidrogenase/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Microfluídica/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors ß-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located â¼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.
Assuntos
Glucosefosfato Desidrogenase , NADP , Domínio Catalítico/genética , Microscopia Crioeletrônica , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/tratamento farmacológico , Deficiência de Glucosefosfato Desidrogenase/enzimologia , Humanos , Mutação , NADP/química , Multimerização ProteicaRESUMO
The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Glutamina/metabolismo , Melanoma/metabolismo , Estresse Oxidativo/fisiologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , NADP/metabolismo , Oxirredução , Via de Pentose Fosfato/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Malaria transmission modelling has demonstrated the potential impact of semiquantitative glucose-6-phosphate dehydrogenase (G6PD) testing and treatment with single-dose tafenoquine for Plasmodium vivax radical cure but has not investigated the associated costs. This study evaluated the cost-effectiveness of P. vivax treatment with tafenoquine after G6PD testing using a transmission model. METHODS AND FINDINGS: We explored the cost-effectiveness of using tafenoquine after G6PD screening as compared to usual practice (7-day low-dose primaquine (0.5 mg/kg/day) without G6PD screening) in Brazil using a 10-year time horizon with 5% discounting considering 4 scenarios: (1) tafenoquine for adults only assuming 66.7% primaquine treatment adherence; (2) tafenoquine for adults and children aged >2 years assuming 66.7% primaquine adherence; (3) tafenoquine for adults only assuming 90% primaquine adherence; and (4) tafenoquine for adults only assuming 30% primaquine adherence. The incremental cost-effectiveness ratios (ICERs) were estimated by dividing the incremental costs by the disability-adjusted life years (DALYs) averted. These were compared to a willingness to pay (WTP) threshold of US$7,800 for Brazil, and one-way and probabilistic sensitivity analyses were performed. All 4 scenarios were cost-effective in the base case analysis using this WTP threshold with ICERs ranging from US$154 to US$1,836. One-way sensitivity analyses showed that the results were most sensitive to severity and mortality due to vivax malaria, the lifetime and number of semiquantitative G6PD analysers needed, cost per malaria episode and per G6PD test strips, and life expectancy. All scenarios had a 100% likelihood of being cost-effective at the WTP threshold. The main limitations of this study are due to parameter uncertainty around our cost estimates for low transmission settings, the costs of G6PD screening, and the severity of vivax malaria. CONCLUSIONS: In our modelling study that incorporated impact on transmission, tafenoquine prescribed after a semiquantitative G6PD testing was highly likely to be cost-effective in Brazil. These results demonstrate the potential health and economic importance of ensuring safe and effective radical cure.
Assuntos
Malária Vivax , Primaquina , Adulto , Criança , Humanos , Primaquina/efeitos adversos , Malária Vivax/diagnóstico , Malária Vivax/tratamento farmacológico , Brasil , Análise de Custo-Efetividade , Glucosefosfato DesidrogenaseRESUMO
Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.
Assuntos
Óxido Nítrico , Nostoc , Nostoc/metabolismo , Nostoc/enzimologia , Nostoc/genética , Óxido Nítrico/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.
Assuntos
Glucosefosfato Desidrogenase , Pressão , Ativação Enzimática , Gluconatos/metabolismo , Gluconatos/química , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/química , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/química , Cinética , Lactonas/química , Lactonas/metabolismo , NADP/metabolismo , NADP/química , TemperaturaRESUMO
BACKGROUND: G6PD deficiency is a common inherited disorder worldwide and has a higher incidence rate in southern China. Many variants of G6PD result from point mutations in the G6PD gene, leading to decreased enzyme activity. This study aimed to analyse the genotypic and phenotypic characteristics of G6PD deficiency in Guangzhou, China. METHODS: In this study, a total of 20,208 unrelated participants were screened from 2020 to 2022. G6PD deficiency was further analysed by quantitative enzymatic assay and G6PD mutation analysis. The unidentified genotype of the participants was further ascertained by direct DNA sequencing. RESULTS: A total of 12 G6PD mutations were identified. Canton (c.1376G>T) and Kaiping (c.1388G>A) were the most common variants, and different mutations led to varying levels of G6PD enzyme activity. Comparing the enzyme activities of the 6 missense mutations between the sexes, we found significant differences (P < 0.05) in the enzyme activities of both male hemizygotes and female heterozygotes. Two previously unreported mutations (c.1438A>T and c.946G>A) were identified. CONCLUSIONS: This study provided detailed genotypes of G6PD deficiency in Guangzhou, which could be valuable for diagnosing and researching G6PD deficiency in this area.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Feminino , Humanos , Masculino , China/epidemiologia , Genótipo , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/genética , Heterozigoto , MutaçãoRESUMO
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.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Glucosefosfato Desidrogenase , Humanos , Camundongos , Animais , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Hemólise , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Antioxidantes , Estudo de Associação Genômica Ampla , Eritrócitos/metabolismo , Doadores de SangueRESUMO
OBJECTIVES: This study aimed to investigate the incidence rate and spectrum of gene mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Huizhou city of southern China to provide a scientific basis for disease prevention and control in the area. METHODS: From March 2003 to December 2022, newborn screening for G6PD enzyme activity was carried out in Huizhou city using the fluorescence quantitative method. Infants who tested positive during the initial screening were diagnosed using the nitroblue tetrazolium ratio method, while a subset of infants received further gene mutation analysis using the multicolor probe melting curve analysis method. RESULTS: A total of 1,291,274 newborns were screened and the screening rate has increased from 20.39% to almost 100%. In the 20-year period, 57,217 (4.43%) infants testing positive during the initial screening. Out of these infants, 49,779 (87%) were recalled for confirmatory testing. G6PD deficiency was confirmed in 39,261 of the recalled infants, indicating a positive predictive value of 78.87%. The estimated incidence rate of G6PD deficiency in the region was 3.49%, which was significantly higher than the average incidence rate of 2.1% in southern China. On the other hand, seven pathogenic G6PD variants were identified in the analysis of the 99 diagnosed infants with the most common being c.1388 G > A (48.5%), followed by c.95 A > G (19.2%), c.1376 G > T (15.2%), c.871 G > A (9.1%), c.1360 C > T (3.0%), c.392 G > T (3.0%), and c.487 G > A (1.0%). CONCLUSION: The incidence of G6PD deficiency in newborns in the Huizhou city was higher than the southern China average level, while the types and frequencies of gene mutations were found to vary slightly from other regions. Our findings suggested that free government screening and nearby diagnosis strategies could reduce the incidence of G6PD deficiency in the area.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Lactente , Humanos , Recém-Nascido , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/genética , Taxa de Mutação , Glucosefosfato Desidrogenase/genética , Mutação , Triagem Neonatal , China/epidemiologiaRESUMO
BACKGROUND: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. METHODS: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. RESULTS: Based on phenotypic testing, the prevalence of G6PD deficiency (< 30% activity) was 6.13% (25/408) and intermediate deficiency (30-70% activity) was found in 15.20% (62/408) of participants. Several G6PD genotypes with newly discovered double missense variants were identified by HRM assays, including G6PD Gaohe + Viangchan, G6PD Valladolid + Viangchan and G6PD Canton + Viangchan. A significantly high frequency of synonymous (c.1311C>T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. CONCLUSIONS: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Malária , Humanos , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Tailândia/epidemiologia , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/análise , Malária/epidemiologia , Aminoquinolinas/efeitos adversosRESUMO
BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.
Assuntos
Antimaláricos , Glucosefosfato Desidrogenase , Malária Vivax , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Cloroquina/uso terapêutico , Guiana Francesa/epidemiologia , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/complicações , Cinética , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/fisiologia , Primaquina/uso terapêutico , Estudos Retrospectivos , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: Cambodia aims to eliminate all forms of malaria by 2025. In 2020, 90% of all malaria cases were Plasmodium vivax. Thus, preventing P. vivax and relapse malaria is a top priority for elimination. 14-day primaquine, a World Health Organization-recommended radical cure treatment regimen, specifically targets dormant hypnozoites in the liver to prevent relapse. Cambodia introduced P. vivax radical cure with primaquine after glucose-6-phosphate dehydrogenase (G6PD) qualitative testing in 2019. This paper presents Cambodia's radical cure Phase I implementation results and assesses the safety, effectiveness, and feasibility of the programme prior to nationwide scale up. METHODS: Phase I implementation was carried out in 88 select health facilities (HFs) across four provinces. Males over 20kgs with confirmed P. vivax or mixed (P. vivax and Plasmodium falciparum) infections were enrolled. A descriptive analysis evaluated the following: successful referral to health facilities, G6PD testing results, and self-reported 14-day treatment adherence. P. vivax incidence was compared before and after radical cure rollout and a controlled interrupted time series analysis compared the estimated relapse rate between implementation and non-implementation provinces before and after radical cure. RESULTS: In the 4 provinces from November 2019 to December 2020, 3,239 P. vivax/mixed infections were reported, 1,282 patients underwent G6PD deficiency testing, and 959 patients received radical cure, achieving 29.6% radical cure coverage among all P. vivax/mixed cases and 98.8% coverage among G6PD normal patients. Among those who initiated radical cure, 747 patients (78%) completed treatment. Six patients reported side effects. In implementation provinces, an average 31.8 relapse cases per month were estimated signaling a 90% (286 cases) reduction in relapse compared to what would be expected if radical cure was not implemented. CONCLUSIONS: Plasmodium vivax radical cure is a crucial tool for malaria elimination in Cambodia. The high coverage of radical cure initiation and adherence among G6PD normal patients demonstrated the high feasibility of providing radical cure at point of care in Cambodia. Incomplete referral from community to HFs and limited capacity of HF staff to conduct G6PD testing in high burden areas led to lower coverage of G6PD testing. Phase I implementation informed approaches to improve referral completion and patient adherence during the nationwide expansion of radical cure in 2021.
Assuntos
Antimaláricos , Deficiência de Glucosefosfato Desidrogenase , Malária Vivax , Malária , Masculino , Humanos , Malária Vivax/tratamento farmacológico , Malária Vivax/epidemiologia , Malária Vivax/prevenção & controle , Primaquina/uso terapêutico , Antimaláricos/uso terapêutico , Glucosefosfato Desidrogenase , Camboja/epidemiologia , Malária/tratamento farmacológico , Plasmodium vivax , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/tratamento farmacológico , RecidivaRESUMO
BACKGROUND: Klinefelter syndrome is a common sex chromosome abnormality in males, characterized by an extra X chromosome compared to normal males. Glucose-6-phosphate dehydrogenase deficiency (G6PD) is an X-linked incomplete dominant defect disorder. In this study, we reported the unexpected detection of Klinefelter syndrome in a patient with G6PD. METHODS: G6PD enzyme activity was measured by immunoenzyme assay, and genetic analysis was performed using a fluorescent PCR melting curve method (PCR-melting curve). Sex chromosome number abnormalities were detected by multiplex ligation-dependent probe amplification (MLPA). The patient also underwent peripheral blood chromosome karyotype analysis. RESULTS: The patient's G6PD and 6PGD enzyme activities were 21.34 U/L and 22.85 U/L, respectively, and their ratio was below the reference range (0.93). The PCR-melting curve displayed a c.1388 heterozygous mutation in this boy, and the Sanger sequencing provided the same results. MLPA results suggested the presence of approxi-mately two copies of the X-chromosome in the boy. Finally, chromosome karyotype analysis confirmed that the boy had Klinefelter syndrome with a karyotype of 47, XXY. CONCLUSIONS: Klinefelter syndrome was accidentally detected during G6PD genetic analysis in a male. X-chromosomes can interfere with the results of G6PD genetic analysis and should be noted.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Glucosefosfato Desidrogenase , Síndrome de Klinefelter , Humanos , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/diagnóstico , Síndrome de Klinefelter/complicações , Masculino , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Glucosefosfato Desidrogenase/genética , Cariotipagem , Mutação , Testes Genéticos/métodos , Cromossomos Humanos X/genéticaRESUMO
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.