RESUMEN
Membrane lipoproteins serve as primary pro-inflammatory virulence factors in Mycoplasma genitalium. Membrane lipoproteins primarily induce inflammatory responses by activating Toll-like Receptor 2 (TLR2); however, the role of the metabolic status of urethral epithelial cells in inflammatory response remains unclear. In this study, we found that treatment of uroepithelial cell lines with M. genitalium membrane lipoprotein induced metabolic reprogramming, characterized by increased aerobic glycolysis, decreased oxidative phosphorylation, and increased production of the metabolic intermediates acetyl-CoA and malonyl-CoA. The metabolic shift induced by membrane lipoproteins is reversible upon blocking MyD88 and TRAM. Malonyl-CoA induces malonylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and malonylated GAPDH could dissociate from the 3' untranslated region of TNF-α and IFN-γ mRNA. This dissociation greatly reduces the inhibitory effect on the translation of TNF-α and IFN-γ mRNA, thus achieving fine-tuning control over cytokine secretion. These findings suggest that GAPDH malonylation following M. genitalium infection is an important inflammatory signal that plays a crucial role in urogenital inflammatory diseases.
Asunto(s)
Citocinas , Células Epiteliales , Interferón gamma , Mycoplasma genitalium , Factor de Necrosis Tumoral alfa , Mycoplasma genitalium/metabolismo , Mycoplasma genitalium/genética , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interferón gamma/metabolismo , Línea Celular , Lipoproteínas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Uretra/microbiología , Uretra/metabolismo , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Factores de Virulencia/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Glucólisis , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genéticaRESUMEN
Macrophage metabolism dysregulation, which is exacerbated by persistent stimulation in infectious and inflammatory diseases, such as diabetic infectious bone defects (DIBD), eventually leads to the failure of bone repair. Here, we have developed an injectable, macrophage-modulated GAPDH-Silence drug delivery system. This microsphere comprises chondroitin sulfate methacrylate (CM) and methacrylated gelatin (GM), while the dimethyl fumarate (DMF)-loaded liposome (D-lip) is encapsulated within the microsphere (CM@GM), named D-lip/CM@GM. Triggered by the over-expressed collagenase in DIBD, the microspheres degrade and release the encapsulated D-lip. D-lip could modulate metabolism by inhibiting GAPDH, which suppresses the over-activation of glycolysis, thus preventing the inflammatory response of macrophages in vitro. While beneficial for macrophages, D-lip/CM@GM is harmful to bacteria. GAPDH, while crucial for glycolysis of staphylococcal species (S. aureus), can be effectively countered by D-lip/CM@GM. We are utilizing existing drugs in innovative ways to target central metabolism for effective eradication of bacteria. In the DIBD model, our results confirmed that the D-lip/CM@GM enhanced bacteria clearance and reprogrammed dysregulated metabolism, thereby significantly improving bone regeneration. In conclusion, this GAPDH-Silence microsphere system may provide a viable strategy to promote diabetic infection bone regeneration.
Asunto(s)
Regeneración Ósea , Macrófagos , Microesferas , Staphylococcus aureus , Animales , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Regeneración Ósea/efectos de los fármacos , Células RAW 264.7 , Staphylococcus aureus/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Masculino , Glucólisis/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Complicaciones de la Diabetes/tratamiento farmacológico , Liposomas/química , Antibacterianos/farmacologíaRESUMEN
Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and Strep-tag affinity proteomics to identify interaction partners of tensin-2 in Flp-In 293 T-REx cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in Flp-In 293 T-REx and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.
Asunto(s)
Glucólisis , Tensinas , Humanos , Tensinas/metabolismo , Fosforilación , Adhesión Celular , Células HEK293 , Unión Proteica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Adhesiones Focales/metabolismo , Proteómica/métodos , Animales , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismoRESUMEN
Salivary proteins of insect herbivores can suppress plant defenses, but the roles of many remain elusive. One such protein is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the saliva of the Recilia dorsalis (RdGAPDH) leafhopper, which is known to transmit rice gall dwarf virus (RGDV). Here we show that RdGAPDH was loaded into exosomes and released from salivary glands into the rice phloem through an exosomal pathway as R. dorsalis fed. In infected salivary glands of R. dorsalis, the virus upregulated the accumulation and subsequent release of exosomal RdGAPDH into the phloem. Once released, RdGAPDH consumed H2O2 in rice plants owing to its -SH groups reacting with H2O2. This reduction in H2O2 of rice plant facilitated R. dorsalis feeding and consequently promoted RGDV transmission. However, overoxidation of RdGAPDH could cause potential irreversible cytotoxicity to rice plants. In response, rice launched emergency defense by utilizing glutathione to S-glutathionylate the oxidization products of RdGAPDH. This process counteracts the potential cellular damage from RdGAPDH overoxidation, helping plant to maintain a normal phenotype. Additionally, salivary GAPDHs from other hemipterans vectors similarly suppressed H2O2 burst in plants. We propose a strategy by which plant viruses exploit insect salivary proteins to modulate plant defenses, thus enabling sustainable insect feeding and facilitating viral transmission.
Asunto(s)
Hemípteros , Peróxido de Hidrógeno , Oryza , Enfermedades de las Plantas , Saliva , Animales , Hemípteros/virología , Peróxido de Hidrógeno/metabolismo , Oryza/virología , Oryza/metabolismo , Enfermedades de las Plantas/virología , Saliva/metabolismo , Saliva/virología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glándulas Salivales/virología , Glándulas Salivales/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Insectos Vectores/virología , Floema/virología , Floema/metabolismo , Reoviridae/fisiología , Glutatión/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Virus de Plantas/fisiología , Defensa de la Planta contra la HerbivoriaRESUMEN
Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.
Asunto(s)
Arginina , Heparina , ARN Interferente Pequeño , Transfección , Humanos , Heparina/farmacología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/genética , Transfección/métodos , Arginina/análogos & derivados , Arginina/farmacología , Hirudinas/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Fragmentos de Péptidos/farmacología , Ácidos Pipecólicos/farmacología , Sulfonamidas/farmacología , Microglobulina beta-2/genética , Antitrombinas/farmacología , Técnicas de Silenciamiento del Gen/métodos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Péptidos de Penetración Celular/farmacología , Terapia Genética/métodos , Anticoagulantes/farmacología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacosRESUMEN
Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.
Asunto(s)
Lenguado , Gliceraldehído-3-Fosfato Deshidrogenasas , Moco , Saccharomyces cerevisiae , Piel , Animales , Lenguado/microbiología , Lenguado/metabolismo , Piel/microbiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Saccharomyces cerevisiae/metabolismo , Moco/metabolismo , Moco/microbiología , Aglutinación , Secuencia de Aminoácidos , Proteínas de Peces/metabolismo , Proteínas de Peces/inmunología , Cromatografía de AfinidadRESUMEN
One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aß) and chronic inflammation in the brain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is easily oxidized under oxidative stress. Numerous data indicate that oxidative modifications of GAPDH in vitro and in cell cultures stimulate GAPDH denaturation and aggregation, and the catalytic cysteine residue Cys152 is important for these processes. Both intracellular and extracellular GAPDH aggregates are toxic for the cells. Interaction of denatured GAPDH with soluble Aß results in mixed insoluble aggregates with increased toxicity. The above-described properties of GAPDH (sensitivity to oxidation and propensity to form aggregates, including mixed aggregates with Aß) determine its role in the pathogenesis of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Gliceraldehído-3-Fosfato Deshidrogenasas , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Humanos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo , Animales , Oxidación-ReducciónRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitous enzyme essential for carbon and energy metabolism in most organisms. Despite its primary role in sugar metabolism, GAPDH is recognized for its involvement in diverse cellular processes, being considered a paradigm among multifunctional/moonlighting proteins. Besides its canonical cytoplasmic location, GAPDH has been detected on cell surfaces or as a secreted protein in prokaryotes, yet little is known about its possible roles in plant symbiotic bacteria. Here we report that Rhizobium etli, a nitrogen-fixing symbiont of common beans, carries a single gap gene responsible for both GAPDH glycolytic and gluconeogenic activities. An active Gap protein is required throughout all stages of the symbiosis between R. etli and its host plant Phaseolus vulgaris. Both glycolytic and gluconeogenic Gap metabolic activities likely contribute to bacterial fitness during early and intermediate stages of the interaction, whereas GAPDH gluconeogenic activity seems critical for nodule invasion and nitrogen fixation. Although the R. etli Gap protein is secreted in a c-di-GMP related manner, no involvement of the R. etli gap gene in c-di-GMP related phenotypes, such as flocculation, biofilm formation or EPS production, was observed. Notably, the R. etli gap gene fully complemented a double gap1/gap2 mutant of Pseudomonas syringae for free life growth, albeit only partially in planta, suggesting potential specific roles for each type of Gap protein. Nevertheless, further research is required to unravel additional functions of the R. etli Gap protein beyond its essential metabolic roles.
Asunto(s)
Phaseolus , Rhizobium etli , Simbiosis , Phaseolus/microbiología , Rhizobium etli/genética , Rhizobium etli/metabolismo , Rhizobium etli/fisiología , Rhizobium etli/crecimiento & desarrollo , Fijación del Nitrógeno , Gluconeogénesis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glucólisis , Nódulos de las Raíces de las Plantas/microbiología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismoRESUMEN
Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.
Asunto(s)
Glucólisis , Infecciones por VIH , VIH-1 , Replicación Viral , Humanos , VIH-1/fisiología , Glucólisis/fisiología , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismoRESUMEN
The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Biomasa , Reactores Biológicos , Glicerol , beta-Fructofuranosidasa , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo , Reactores Biológicos/microbiología , Glicerol/metabolismo , Fermentación , Aspergillus niger/genética , Aspergillus niger/enzimología , Saccharomycetales/genética , Saccharomycetales/enzimología , Oxígeno/metabolismo , Regiones Promotoras Genéticas , Medios de Cultivo/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , OligosacáridosRESUMEN
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.
Asunto(s)
Células Epiteliales , Oxidación-Reducción , Estrés Oxidativo , Compuestos de Sulfhidrilo , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Estrés Oxidativo/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hemiterpenos/metabolismo , Peróxidos/metabolismoRESUMEN
AIM: Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. METHODS: We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. RESULTS: We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. CONCLUSIONS: When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.
Asunto(s)
Proteína Ácida Fibrilar de la Glía , Bancos de Tejidos , Humanos , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adulto , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Encéfalo/metabolismo , Corteza Prefrontal/metabolismo , Lóbulo Temporal/metabolismoRESUMEN
Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.
Asunto(s)
Péptidos Antimicrobianos , Membrana Celular , Proteínas de Peces , Animales , Secuencia de Aminoácidos , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Proteínas de Peces/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Simulación de Dinámica Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial proapoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N,N,N',N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a proapoptotic protein for VDAC-gating regulation and cell death initiation during seed aging.
Asunto(s)
Muerte Celular , Glutatión , Semillas , Semillas/metabolismo , Glutatión/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glucólisis , Plantas Modificadas Genéticamente , Zinc/metabolismoRESUMEN
The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.
Asunto(s)
Candida , Candidiasis , Inmunoglobulina G , Animales , Ratones , Candida/inmunología , Candida/patogenicidad , Humanos , Candidiasis/inmunología , Candidiasis/microbiología , Candidiasis/sangre , Inmunoglobulina G/sangre , Antígenos Fúngicos/inmunología , Antígenos Fúngicos/sangre , Proteómica/métodos , Candida albicans/inmunología , Candida albicans/patogenicidad , Proteínas Fúngicas/inmunología , Fosfoglicerato Mutasa/inmunología , Fosfoglicerato Quinasa/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/inmunología , Femenino , VirulenciaRESUMEN
OBJECTIVES: This study aimed to discover novel antifungals targeting Candida albicans glyceraldehyde-3-phosphate dehydrogenase (CaGAPDH), have an insight into inhibitory mode, and provide evidence supporting CaGAPDH as a target for new antifungals. METHODS: Virtual screening was utilized to discover inhibitors of CaGAPDH. The inhibitory effect on cellular GAPDH was evaluated by determining the levels of ATP, NAD, NADH, etc., as well as examining GAPDH mRNA and protein expression. The role of GAPDH inhibition in C. albicans was supported by drug affinity responsive target stability and overexpression experiments. The mechanism of CaGAPDH inhibition was elucidated by Michaelis-Menten enzyme kinetics and site-specific mutagenesis based on docking. Chemical synthesis was used to produce an improved candidate. Different sources of GAPDH were used to evaluate inhibitory selectivity across species. In vitro and in vivo antifungal tests, along with anti-biofilm activity, were carried out to evaluate antifungal potential of GAPDH inhibitors. RESULTS: A natural xanthone was identified as the first competitive inhibitor of CaGAPDH. It demonstrated in vitro anti-C. albicans potential but also caused hemolysis. XP-W, a synthetic side-chain-optimized xanthone, demonstrated a better safety profile, exhibiting a 50-fold selectivity for CaGAPDH over human GAPDH. XP-W also exhibited potent anti-biofilm activity and displayed broad-spectrum anti-Candida activities in vitro and in vivo, including multi-azole-resistant C. albicans. CONCLUSIONS: These results demonstrate for the first time that CaGAPDH is a valuable target for antifungal drug discovery, and XP-W provides a promising lead.
Asunto(s)
Antifúngicos , Candida albicans , Gliceraldehído-3-Fosfato Deshidrogenasas , Xantonas , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Xantonas/farmacología , Xantonas/química , Antifúngicos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Animales , Biopelículas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Humanos , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Simulación del Acoplamiento Molecular , Inhibidores Enzimáticos/farmacología , Ratones , Descubrimiento de DrogasRESUMEN
Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.
Asunto(s)
Virus del Dengue , Dengue , Humanos , Virus del Dengue/fisiología , Proteómica , NAD/metabolismo , Hepatocitos/metabolismo , Glucólisis , Hígado/metabolismo , Replicación Viral , Proteoma/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismoRESUMEN
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.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Peróxido de Hidrógeno/farmacología , Gliceraldehído 3-Fosfato , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Oxidación-Reducción , Ácido Láctico/uso terapéuticoRESUMEN
INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.
Asunto(s)
COVID-19 , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante) , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/fisiología , COVID-19/virología , Células HEK293 , Betacoronavirus/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Neumonía Viral/virología , Neumonía Viral/inmunología , Pandemias , Infecciones por Coronavirus/virología , Enzima Convertidora de Angiotensina 2/metabolismoRESUMEN
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.