Beyond Glucose: The Crucial Role of Redox Signaling in β-Cell Metabolic Adaptation
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ABSTRACT:
PROVIDER: PRJNA1148670 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30260691.fastq.gz | Fastqsanger.gz | |||
| SRR30260692.fastq.gz | Fastqsanger.gz | |||
| SRR30260693.fastq.gz | Fastqsanger.gz | |||
| SRR30260694.fastq.gz | Fastqsanger.gz | |||
| SRR30260695.fastq.gz | Fastqsanger.gz |
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Project description:Objective: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. Methods: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. Results: Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. Conclusions: Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.
2024-09-15 | GSE274980 | GEO
Project description:Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells.
2024-09-30 | PXD052765 | Pride
Project description:The faecal indicator bacterium Escherichia coli K12 was used to study the cellular events that take place at the transcription level using the microarray technology during short-term (physiological) and long-term (genetic) adaptation to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 hours of glucose-limited continuous culture at a dilution rate of 0.3 h-1 with those from batch culture with glucose excess. Keywords: glucose-limited continuous culture, adaptation, microarray, high affinity transport systems, transcriptome, Escherichia coli
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Project description:The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress an significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulfide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potentials of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term adaptation of the cells from normoxic to hypoxic conditions.
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Project description:Intracellular sugar compartmentation is critically involved in plant development and acclimation to challenging environmental conditions. Sugar transport proteins are present in the plasma membrane and in membranes of organelles such as vacuoles, the Golgi apparatus and plastids. Here we report on a novel transporter of the Monosaccharide Transporter Family (MSF), that is the closest phylogenetic homolog to the chloroplast-localized glucose transporter pGlcT and that we therefore term plastidic glucose transporter 2 (pGlcT2). Its gene complemented glucose uptake deficiency of an Escherichia coli ptsG/manXYZ mutant strain and biochemical characterization revealed that this transporter specifically facilitates glucose transport, whereas other sugars do not serve as substrates. pGlcT2-GFP fusions located to the chloroplast envelope, and promoter activity analyses indicated that pGlcT2 is mainly produced in seedlings and in the rosette center of mature Arabidopsis plants. In the absence of additional sugars, pGlcT2-overexpression lines exhibited impaired germination. In conjunction with molecular and metabolic data we propose that pGlcT2 acts as a glucose importer that can limit cytosolic glucose availability in developing pGlcT2-overexpressing seedlings. Overexpression, as well as deletion of pGlcT2 resulted in impaired growth efficiency under long day and especially under continuous light conditions, suggesting that pGlcT2 contributes to a release of glucose derived from starch mobilization late in the light phase. These data thus indicate that the facilitator pGlcT2 changes its direction of glucose transport during plant development and suggest that the activity of pGlcT2 must be controlled spatially and temporarily, in order to prevent developmental defects during adaptation to periods of extended light.
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Project description:Glucose is a widely used carbon source in laboratory practice to culture Aspergillus fumigatus, however, glucose availability is often low in its “natural habitats” including the human body. We used a physiological–transcriptomical approach to reveal differences between A. fumigatus Af293 cultures incubated on glucose, glucose and peptone, peptone (carbon limitation), or without any carbon source (carbon starvation). Autolytic cell wall degradation was upregulated by both carbon starvation and limitation. The importance of autolytic cell wall degradation in adaptation to carbon stress was also highlighted by approximately 12.4% of the A. fumigatus genomes harbor duplication of genes involved in N-acetyl glucosamine utilization. Glucose withdrawal increased redox imbalance, altered both the transcription of antioxidative enzyme genes and oxidative stress tolerance, downregulated iron acquisition, but upregulated heme protein genes. Transcriptional activity of the Gliotoxin cluster was low in all experiments, while the Fumagillin cluster showed substantial activity both on glucose and under carbon starvation, and the Hexadehydro-astechrome cluster only on glucose. We concluded that glucose withdrawal substantially modified the physiology of A. fumigatus including processes that contribute to virulence. This may explain the challenge of predicting the in vivo behavior of A. fumigatus based on data from glucose rich cultures.
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Project description:Thiol-dependent redox regulation is essential for the rapid adaptation of chloroplast metabolism to unpredictable changes of light intensity. The disulfide reductase activity of thioredoxins (Trxs), which relies on photo-reduced ferredoxin (Fdx) and a Fdx-dependent Trx reductase (FTR), constitutes the Fdx-FTR-Trxs system, which links chloroplast redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase (NTR) with a joint Trx domain, NTRC. The activity of these two redox systems is integrated by the balance of the hydrogen peroxide scavenging enzyme 2-Cys peroxiredoxin (2-Cys Prx), which thus plays a key role in maintaining the reducing capacity of chloroplast Trxs in response to light intensity. Based on the severe phenotype of mutant lines lacking NTRC, it is clear that this enzyme plays an essential role in chloroplast redox homeostasis. However, whether the function of NTRC depends on its capacity of reduce 2-Cys Prxs or has additional targets remains unknown. Here, we have addressed this issue by a comparative analysis of the triple mutant of Arabidopsis thaliana, ntrc-2cpab, simultaneously lacking 2-Cys Prxs and NTRC, and the double mutant 2cpab lacking 2-Cys Prxs. The phenotype of the ntrc-2cpab mutant is indistinguishable of the 2cpab mutant, as shown by growth rate, photosynthesis performance, light-dependent redox regulation of enzyme activity and comparative transcriptomics based RNA-Seq. Based on these results, we propose that the function of NTRC in chloroplast redox homeostasis is exerted by the regulation of the redox balance of 2-Cys Prxs rather than by the direct reduction of additional targets.
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Project description:The faecal indicator bacterium Escherichia coli K12 was used to study the cellular events that take place at the transcription level using the microarray technology during short-term (physiological) and long-term (genetic) adaptation to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 hours of glucose-limited continuous culture at a dilution rate of 0.3 h-1 with those from batch culture with glucose excess. Wild-type Escherichia coli K12 MG 1655 (ATCC 700926) was grown at 37 °C in mineral medium. Growth was followed by measuring OD546 in regular intervals to confirm that cells were growing at ï?max (0.63 ± 0.02 h-1). A sample for transcriptome analysis was taken from the batch exponential phase at an OD546 of ï?¾ 0.4, and immediately after sampling the medium flow was started for continuous cultivation with a dilution rate of 0.3 h-1. Steady-state with respect to biomass and residual glucose concentrations was reached approximately 15 hours after this manipulation. 40 and 500 hours after switching to the glucose-limited continuous culture mode 800 ml of culture liquid was withdrawn from the chemostat, immediately cooled down on crushed ice and cells were collected by centrifugation at 4 °C. Total RNA from E. coli cells was isolated as described earlier (Sambrook et al., 1989). RNA in samples was quantified spectrophotometrically by measuring extinction at 260 nm and purity (free from DNA) was checked by gel electrophoresis. Synthesis of cDNA from RNA was performed with the CyScribe First-Strand cDNA Labelling Kit (Amersham Bioscience, Little Chalfont, England). The purified cDNA was concentrated to 5 μl and was mixed with 120 μl of hybridization buffer (MWG-Biotech AG), heated to 95 °C for 3 min and cooled down on ice for 3 min. The hybridization mixture was then added to the microarray slide and covered with a coverslip. The hybridisation slide was incubated over night at 42 °C. After the hybridization step the slide was washed three times, the first time for 5 min in 2x (times concentrated) SSC - 0.1 % SDS, the second time for 5 min in 1x SSC, and finally for 5 min in 0.1x SSC. SSC buffer was prepared as 20x solution containing 0.3M Na-citrate and 3M NaCl at pH 7.0. The slides were dried by centrifugation at room temperature for 2 min at 500g. Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, USA). Induction factors were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the induction factors of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Data from the independent experiments were combined, genes that were differentially regulated â?¥ 2 and â?¤ 0.5 (t-test, p â?¤ 0.05) were defined as being statistically significant.
2010-07-01 | E-GEOD-4706 | biostudies-arrayexpress