The glutathione system is essential to maintain the thiol redox balance in the mitochondria, but not in the cytosol, of fission yeast
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE283698 | GEO | 2025/04/10
REPOSITORIES: GEO
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Project description:The glutathione system is essential to maintain the thiol redox balance in the mitochondria, but not in the cytosol, of fission yeast
| PRJNA1195057 | ENA
Project description:We systematically examined transcription and RNA-processing in mitochondria of the petite-negative fission yeast Schizosaccharomyces pombe. Two presumptive transcription initiation sites at opposite positions on the circular-mapping mtDNA were confirmed by in vitro capping of primary transcripts with guanylyl-transferase. The major promoter (Pma) is located adjacent to the 5'-end of the rnl gene, and a second, minor promoter (Pmi) upstream from cox3. The primary 5'-termini of the mature rnl and cox3 transcripts remain unmodified. A third predicted accessory transcription initiation site is within the group IIA1 intron of the cob gene (cobI1). The consensus promoter motif of S. pombe closely resembles the nonanucleotide promoter motifs of various yeast mtDNAs. We further characterized all mRNAs and the two ribosomal RNAs by Northern hybridization, and precisely mapped their 5'- and 3'-ends. The mRNAs have leader sequences with a length of 38 up to 220 nt and, in most instances, are created by removal of tRNAs from large precursor RNAs. Like cox2 and rnl, cox1 and cox3 are not separated by tRNA genes; instead, transcription initiation from the promoters upstream from rnl and cox3 compensates for the lack of tRNA-mediated 5'-processing. The 3'-termini of mRNAs and of SSU rRNA are processed at distinct, C-rich motifs that are located at a variable distance (1-15 nt) downstream from mRNA and SSU-rRNA coding regions. The accuracy of RNA-processing at these sites is sequence-dependent. Similar 3'-RNA-processing motifs are present in species of the genus Schizosaccharomyces, but not in budding yeasts that have functionally analogous A+T-rich dodecamer processing signals.
| S-EPMC1370763 | biostudies-literature
Project description:BackgroundNADPH is an essential co-factor supporting the function of enzymes that participate in both inflammatory and anti-inflammatory pathways in myeloid cells, particularly macrophages. Although individual NADPH-dependent pathways are well characterized, how these opposing pathways are co-regulated to orchestrate an optimized inflammatory response is not well understood. To investigate this, techniques to track the consumption of NADPH need to be applied. Deuterium tracing of NADPH remains the gold standard in the field, yet this setup of mass-spectrometry is technically challenging and not readily available to most research groups. Furthermore, NADPH pools are compartmentalized in various organelles with no known membrane transporters, suggesting that NADPH-dependent pathways are regulated in an organelle-specific manner. Conventional methods such as commercial kits are limited to quantifying NADPH in whole cells and not at the resolution of specific organelles. These limitations reflect the need for a novel assay that can readily measure the consumption rate of NADPH in different organelles.MethodsWe devised an assay that measures the consumption rate of NADPH by glutathione-disulfide reductase (GSR) in the mitochondria and the cytosol of RAW264.7 macrophage cell lines. RAW264.7 cells were transfected with Apollo-NADP+ sensors targeted to the mitochondria or the cytosol, followed by the treatment of 2-deoxyglucose and diamide. Intravital imaging over time then determined GSR-dependent NADPH consumption in an organelle-specific manner.DiscussionIn lipopolysaccharide (LPS)-stimulated RAW264.7 cells, cytosolic and mitochondrial NADPH was consumed by GSR in a time-dependent manner. This finding was cross validated with a commercially available NADPH kit that detects NADPH in whole cells. Loading of RAW264.7 cells with oxidized low-density lipoprotein followed by LPS stimulation elevated GSR expression, and this correlated with a more rapid drop in cytosolic and mitochondrial NADPH in our assay. The current limitation of our assay is applicability to transfectable cell lines, and higher expression of plasmid-encoded sensors relative to endogenous glucose-6-phosphate dehydrogenase.
| S-EPMC11620681 | biostudies-literature
Project description:Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.
| S-EPMC9686363 | biostudies-literature
Project description:In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid β-oxidation. During this process, NAD+ is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD+ by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the cytosol by the malate/oxaloacetate shuttle. The ultimate step in lysine biosynthesis, the NAD+-dependent dehydrogenation of saccharopine to lysine, is another NAD+-dependent reaction performed inside peroxisomes. We have found that in glucose grown cells, both the malate/oxaloacetate shuttle and a glycerol-3-phosphate dehydrogenase 1(Gpd1p)-dependent shuttle are able to maintain the intraperoxisomal redox balance. Single mutants in MDH3 or GPD1 grow on lysine-deficient medium, but an mdh3/gpd1Δ double mutant accumulates saccharopine and displays lysine bradytrophy. Lysine biosynthesis is restored when saccharopine dehydrogenase is mislocalised to the cytosol in mdh3/gpd1Δ cells. We conclude that the availability of intraperoxisomal NAD+ required for saccharopine dehydrogenase activity can be sustained by both shuttles. The extent to which each of these shuttles contributes to the intraperoxisomal redox balance may depend on the growth medium. We propose that the presence of multiple peroxisomal redox shuttles allows eukaryotic cells to maintain the peroxisomal redox status under different metabolic conditions.
| S-EPMC5605654 | 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:Lateral diffusion barriers compartmentalize membranes to generate polarity or asymmetrically partition membrane-associated macromolecules. Budding yeasts assemble such barriers in the endoplasmic reticulum (ER) and the outer nuclear envelope at the bud neck to retain aging factors in the mother cell and generate naïve and rejuvenated daughter cells. However, little is known about whether other organelles are similarly compartmentalized. Here, we show that the membranes of mitochondria are laterally compartmentalized at the bud neck and near the cell poles. The barriers in the inner mitochondrial membrane are constitutive, whereas those in the outer membrane form in response to stresses. The strength of mitochondrial diffusion barriers is regulated positively by spatial cues from the septin axis and negatively by retrograde (RTG) signaling. These data indicate that mitochondria are compartmentalized in a fission-independent manner. We propose that these diffusion barriers promote mitochondrial polarity and contribute to mitochondrial quality control.
| S-EPMC10783438 | biostudies-literature
Project description:Thiamine (vitamin B1), under the proper conditions, is able to reversibly open the thiazole ring, forming a thiol-bearing molecule that can be further oxidized to the corresponding disulfide. To improve the bioavailability of the vitamin, several derivatives of thiamine in the thioester or disulfide form were developed and extensively studied over time, as apparent from the literature. We have examined three thiamine-derived disulfides: thiamine disulfide, sulbutiamine, and fursultiamine with reference to their intervention in modulating the thiol redox state. First, we observed that both glutathione and thioredoxin (Trx) systems were able to reduce the three disulfides. In particular, thioredoxin reductase (TrxR) reduced these disulfides either directly or in the presence of Trx. In Caco-2 cells, the thiamine disulfide derivatives did not modify the total thiol content, which, however, was significantly decreased by the concomitant inhibition of TrxR. When oxidative stress was induced by tert-butyl hydroperoxide, the thiamine disulfides exerted a protective effect, indicating that the thiol form deriving from the reduction of the disulfides might be the active species. Further, the thiamine disulfides examined were shown to increase the nuclear levels of the transcription factor nuclear factor erythroid 2 related factor 2 and to stimulate both expression and activity of NAD(P)H quinone dehydrogenase 1 and TrxR. However, other enzymes of the glutathione and Trx systems were scarcely affected. As the thiol redox balance plays a critical role in oxidative stress and inflammation, the information presented can be of interest for further research, considering the potential favorable effect exerted in the cell by many sulfur compounds, including the thiamine-derived disulfides.
| S-EPMC11681303 | biostudies-literature
Project description:What is cause and what is consequence of aging and whether reactive oxygen species (ROS) contribute to this phenomenon is debated since more than 50 years. Notwithstanding, little is known about the cellular buffer and redox systems in aging Saccharomyces cerevisiae, which is a model for aging stem cells. Using genetically encoded fluorescent sensors, we measured pH, H2O2 levels and the glutathione redox potential compartment-specific in the cytosol of living, replicatively aging yeast cells, growing under fermenting and respiratory conditions until the end of their lifespan. We found that the pH decreases under both conditions at later stages of the replicative lifespan. H2O2 levels increase in fermenting cells in the post-replicative stage, but increase continuously with age in respiring cells. The glutathione redox couple becomes also more oxidizing in respiring cells but surprisingly more reducing under fermenting conditions. In strains deleted for the gene encoding glutathione reductase Glr1, such a reduction of the glutathione redox couple with age is not observed. We demonstrate that in vivo Glr1 is activated at lower pH explaining the reduced glutathione potential. The deletion of glr1 dramatically increases the glutathione redox potential especially under respiratory conditions but does not reduce lifespan. Our data demonstrate that pH and the glutathione redox couple is linked through Glr1 and that yeast cells can cope with a high glutathione redox potential without impact on longevity. Our data further suggest that a breakdown of cellular energy metabolism marks the end of replicative lifespan in yeast.
| S-EPMC5515007 | biostudies-literature
Project description:Forkhead Box O (FoxO) transcription factors act in adult stem cells to preserve their regenerative potential. Previously, we reported that FoxO maintains the long-term proliferative capacity of neural stem/progenitor cells (NPCs), and that this occurs, in part, through the maintenance of redox homeostasis. Herein, we demonstrate that among the FoxO3-regulated genes in NPCs are a host of enzymes in central carbon metabolism that act to combat reactive oxygen species (ROS) by directing the flow of glucose and glutamine carbon into defined metabolic pathways. Characterization of the metabolic circuit observed upon loss of FoxO3 revealed a drop in glutaminolysis and filling of the tricarboxylic acid (TCA) cycle. Additionally, we found that glucose uptake, glucose metabolism and oxidative pentose phosphate pathway activity were similarly repressed in the absence of FoxO3. Finally, we demonstrate that impaired glucose and glutamine metabolism compromises the proliferative potential of NPCs and that this is exacerbated following FoxO3 loss. Collectively, our findings show that a FoxO3-dependent metabolic programme supports redox balance and the neurogenic potential of NPCs.
| S-EPMC3791369 | biostudies-literature