Novel Transgenic Lines to Analyze Renal Glutathione Redox Potential In Vivo.
Ontology highlight
ABSTRACT:
SUBMITTER: Sugano Y
PROVIDER: S-EPMC7153637 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundPeroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood.MethodsLive cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H2O2) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH.ResultsExposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H2O2 production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H2O2 or the low molecular weight alkyl tert-butyl hydroperoxide (TBH).ConclusionsLong chain lipid hydroperoxides can directly alter cytosolic EGSH independent of secondary lipid oxidation products or H2O2 production. NADPH has a protective role against 9-HpODE induced EGSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation.SignificanceThese results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.
| S-EPMC6823641 | biostudies-literature
Project description:NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.
| S-EPMC3112490 | biostudies-literature
Project description:Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.
| S-EPMC4294471 | biostudies-literature
Project description:Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (E(GSH)) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with E(GSH)-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of E(GSH) in the IMS, thus explaining a steady-state E(GSH) in the IMS which is similar to the cytosol. Moreover, we show that the local E(GSH) contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells.
| S-EPMC3400016 | biostudies-literature
Project description:Redox status affects various cellular activities, such as proliferation, differentiation, and death. Recent studies suggest pivotal roles of reactive oxygen species not only in pathogenesis under oxidative insult but also in intracellular signal transduction. Glutathione is present in several millimolar concentrations in the cytoplasm and has multiple roles in the regulation of cellular homeostasis. Two enzymes, ?-glutamylcysteine synthetase and glutathione synthetase, constitute the de novo synthesis machinery, while glutathione reductase is involved in the recycling of oxidized glutathione. Multidrug resistant proteins and some other transporters are responsible for exporting oxidized glutathione, glutathione conjugates, and S-nitrosoglutathione. In addition to antioxidation, glutathione is more positively involved in cellular activity via its sulfhydryl moiety of a molecule. Animals in which genes responsible for glutathione metabolism are genetically modified can be used as beneficial and reliable models to elucidate roles of glutathione in vivo. This review article overviews recent progress in works related to genetically modified rodents and advances in the elucidation of glutathione-mediated reactions.
| S-EPMC3171681 | biostudies-literature
Project description:Cysteine is susceptible to a variety of modifications by reactive oxygen and nitrogen oxide species, including glutathionylation; and when two cysteines are involved, disulfide formation. Glutathione-cysteine adducts may be removed from proteins by glutaredoxin, whereas disulfides may be reduced by thioredoxin. Glutaredoxin is homologous to the disulfide-reducing thioredoxin and shares similar binding modes of the protein substrate. The evolution of these systems is not well characterized. When a single Cys is present in a protein, conjugation of the redox buffer glutathione may induce conformational changes, resulting in a simple redox switch that effects a signaling cascade. If a second cysteine is introduced into the sequence, the potential for disulfide formation exists. In favorable protein contexts, a bistable redox switch may be formed. Because of glutaredoxin's similarities to thioredoxin, the mutated protein may be immediately exapted into the thioredoxin-dependent redox cycle upon addition of the second cysteine. Here we searched for examples of protein substrates where the number of redox-active cysteine residues has changed throughout evolution. We focused on cross-strand disulfides (CSDs), the most common type of forbidden disulfide. We searched for proteins where the CSD is present, absent and also found as a single cysteine in protein orthologs. Three different proteins were selected for detailed study-CD4, ERO1, and AKT. We created phylogenetic trees, examining when the CSD residues were mutated during protein evolution. We posit that the primordial cysteine is likely to be the cysteine of the CSD which undergoes nucleophilic attack by thioredoxin. Thus, a redox-active disulfide may be introduced into a protein structure by stepwise mutation of two residues in the native sequence to Cys. By extension, evolutionary acquisition of structural disulfides in proteins can potentially occur via transition through a redox-active disulfide state.
| S-EPMC4354306 | biostudies-literature
Project description:The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.
| S-EPMC8087105 | biostudies-literature
Project description:In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be -314.2±3.1 mV and -313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.
| S-EPMC3857819 | biostudies-literature
Project description:The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant-negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions.
| S-EPMC3197968 | biostudies-literature
Project description:IntroductionThe gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.Materials and methodsIDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.ResultsIn total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.ConclusionsWe have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.
| S-EPMC6023169 | biostudies-literature