scholarly journals Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Antonella De Palma ◽  
Giulia Fanelli ◽  
Elisabetta Cretella ◽  
Veronica De Luca ◽  
Raffaele Antonio Palladino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Consumption ◽  
Redox Balance ◽  
Glycolytic Enzymes ◽  
Glycolytic Flux ◽  
Direct Role ◽  
Content Type ◽  
Proteomic Approach ◽  
Altered Glucose

ABSTRACT Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell. IMPORTANCE We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors.

scholarly journals A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis inS. cerevisiaeMitochondria

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Teresa Anna Giancaspero ◽  
Vittoria Locato ◽  
Maria Barile
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotinamide Adenine Dinucleotide ◽  
Flavin Adenine Dinucleotide ◽  
Redox Balance ◽  
Redox Status ◽  
Cellular Redox ◽  
Isolated Mitochondria ◽  
Nudix Hydrolases ◽  
Order Of Magnitude

Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear inSaccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+and NADH according to a noncompetitive inhibition, withKivalues that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation inS. cerevisiae.

scholarly journals Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption

2018 ◽  
Vol 314 (3) ◽  
pp. E214-E223 ◽  
Author(s):  
Sally Winther ◽  
Marie S. Isidor ◽  
Astrid L. Basse ◽  
Nina Skjoldborg ◽  
Amanda Cheung ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Glucose Consumption ◽  
Glucose Transporters ◽  
Glycolytic Enzymes ◽  
Brown Adipocyte ◽  
Glycolytic Flux ◽  
Acid Oxidation ◽  
Cold Stimulation ◽  
Brown Adipocytes ◽  
Effect Of Glucose

During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.

scholarly journals Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

2011 ◽  
Vol 11 (3) ◽  
pp. 334-342 ◽  
Author(s):  
Dariusz Abramczyk ◽  
Stacey Holden ◽  
Christopher J. Page ◽  
Richard J. Reece
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcriptional Control ◽  
Transcriptional Response ◽  
Content Type ◽  
Considerable Debate ◽  
Active Transcription ◽  
Gal Genes ◽  
The Subject

ABSTRACT The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins—a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)—control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

scholarly journals Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

2014 ◽  
Vol 82 (12) ◽  
pp. 5099-5109 ◽  
Author(s):  
Paula Gaspar ◽  
Firas A. Y. Al-Bayati ◽  
Peter W. Andrew ◽  
Ana Rute Neves ◽  
Hasan Yesilkaya
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactate Production ◽  
Virulence Gene ◽  
Redox Balance ◽  
Central Metabolism ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Key Enzyme

ABSTRACTStreptococcus pneumoniaeis a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenicldhmutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed byin vivonuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of theldhmutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.

scholarly journals Gcn5 and Ubp8 dependent ubiquitylation affects glycolysis in yeast

2019 ◽  
Author(s):  
Antonella De Palma ◽  
Giulia Fanelli ◽  
Elisabetta Cretella ◽  
Veronica De Luca ◽  
Valentina Panzeri ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Human Cells ◽  
Glycolytic Enzymes ◽  
Glycolytic Flux ◽  
Future Research ◽  
Saga Complex ◽  
Epigenetic Factors ◽  
Innovative Strategies

AbstractMany of the molecular mechanisms affected by ubiquitylation are highly conserved from yeast to humans and are associated to a plethora of diseases including cancers. To elucidate the regulatory role of epigenetic factors such as the catalytic subunits of SAGA complex, KAT-Gcn5 and Ub-protease Ubp8, on ubiquitylation of non-histone proteins we have performed a comprehensive analysis of the Ub-proteome in yeastSaccharomyces cerevisiaein strains disrupted in Gcn5, Ubp8 or both respect to wild type. We found significative alteration of ubiquitylation in proteins belonging to different functional categories with a recurrence of identical proteins in absence of Gcn5 or Ubp8 indicating shared targets and their interlaced function. Among the processes involved we noteworthy identified all major enzymes engaged in energy metabolism and glycolysis such as PFK1, PFK2 and others showing increased ubiquitylation respect to WT. We showed that the higher degree of ubiquitylation found is at post-translational level and does not depend on transcription. Noteworthy, we foundin vivosevere defects of growth in poor sugar medium and inability to adaptive switch from fermentative to respiratory growth in strains lacking Gcn5 and Ubp8. Our findings data provide a novel, direct link, between metabolism and epigenetic control with a novel role of DUB-Ubp8 and KAT-Gcn5 on the ubiquitylation marking all the main glycolytic enzymes required for an effective execution of the glycolytic flux. Collectively our experimental results and the proposed model can lead to future research and innovative strategies that by targeting epigenetic modulators might be able to lower sugar utilization also in human cells.Author SummaryMolecular mechanisms dissected in simple yeast might be translated to similar circuitries in human cells for new discoveries in human diseases including cancer. Ubiquitylation of proteins is an evolutionary conserved mechanism required for many biological processes. Different post-translational modifications (PTMs) such as ubiquitylation, acetylation, methylation etc. are reciprocally regulated for deposition or removal. Epigenetic factors writing the PTMs code are often components of multiproteic complexes such as SAGA complex that holds the K-acetyltransferase (KAT) Gcn5 and the Ubiquitin-protease (DUB) Ubp8 highly conserved in Evolution. Cells respond to environment and nutrients by changing metabolism and group of enzymes involved in specific pathways are often coregulated by the deposition of selected PTMs. This study analyses the composition and quantitation of Ub-proteins differentially modified in absence of KAT-Gcn5 and DUB-Ubp8 in yeast. Interstingly, we highlighted the role of Gcn5/Ubp8 dependent ubiquitylation in marking major glycolytic enzymes necessary for glucose utilization. Our study suggests a novel regulatory pathway and, considering that lowering glycolysis is a promising strategy to target tumor metabolism, we propose this study as an interesting perspective to lower enhanced glycolysis in tumors.

scholarly journals Energetics of Inhibition: Insights with a Computational Model of the Human GABAergic Neuron–Astrocyte Cellular Complex

2010 ◽  
Vol 30 (11) ◽  
pp. 1834-1846 ◽  
Author(s):  
Rossana Occhipinti ◽  
Erkki Somersalo ◽  
Daniela Calvetti
Keyword(s):  
Glucose Consumption ◽  
Compartment Model ◽  
Cell Types ◽  
Redox Balance ◽  
Gabaergic Neurons ◽  
Neuronal Uptake ◽  
Activity Levels ◽  
Inhibitory Neurotransmitter ◽  
Metabolic Coupling

We investigate metabolic interactions between astrocytes and GABAergic neurons at steady states corresponding to different activity levels using a six-compartment model and a new methodology based on Bayesian statistics. Many questions about the energetics of inhibition are still waiting for definite answers, including the role of glutamine and lactate effluxed by astrocytes as precursors for γ-aminobutyric acid (GABA), and whether metabolic coupling applies to the inhibitory neurotransmitter GABA. Our identification and quantification of metabolic pathways describing the interaction between GABAergic neurons and astrocytes in connection with the release of GABA makes a contribution to this important problem. Lactate released by astrocytes and its neuronal uptake is found to be coupled with neuronal activity, unlike glucose consumption, suggesting that in astrocytes, the metabolism of GABA does not require increased glycolytic activity. Negligible glutamine trafficking between the two cell types at steady state questions glutamine as a precursor of GABA, not excluding glutamine cycling as a transient dynamic phenomenon, or a prominent role of GABA reuptake. Redox balance is proposed as an explanation for elevated oxidative phosphorylation and adenosine triphosphate hydrolysis in astrocytes, decoupled from energy requirements.

Nitric oxide mediation of chemoregulation but not autoregulation of cerebral blood flow in primates

1996 ◽  
Vol 84 (1) ◽  
pp. 71-78 ◽  
Author(s):  
B. Gregory Thompson ◽  
Ryszard M. Pluta ◽  
Mary E. Girton ◽  
Edward H. Oldfield
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Cerebral Blood Flow ◽  
No Production ◽  
Direct Role ◽  
Content Type ◽  
Continuous Release ◽  
Fine Print

✓ The authors sought to develop a model for assessing in vivo regulation of cerebral vasoregulation by nitric oxide (NO), originally described as endothelial-derived relaxing factor, and to use this model to establish the role of NO in the regulation of cerebral blood flow (CBF) in primates. By using regional intraarterial perfusion, the function of NO in cerebral vasoregulation was examined without producing confounding systemic physiological effects. Issues examined were: whether resting vasomotor tone requires NO; whether NO mediates vasodilation during chemoregulation and autoregulation of CBF; and whether there is a relationship between the degree of hypercapnia and hypotension and NO production. Twelve anesthetized (0.5% isoflurane) cynomolgus monkeys were monitored continuously for cortical CBF, PaCO2, and mean arterial pressure (MAP), which were systematically altered to provide control and experimental curves of chemoregulation (CBF vs. PaCO2) and autoregulation (CBF vs. MAP) during continuous intracarotid infusion of 1) saline and 2) an NO synthase inhibitor (NOSI), either l-n-monomethyl arginine or nitro l-arginine. During basal conditions (PaCO2 of 38–42 mm Hg) NOSI infusion of internal carotid artery (ICA) reduced cortical CBF from 62 (saline) to 53 ml/100 g/per minute (p < 0.01), although there was no effect on MAP. Increased CBF in response to hypercapnia was completely blocked by ICA NOSI. The difference in regional (r)CBF between ICA saline and NOSI infusion increased linearly with PaCO2 when PaCO2 was greater than 40 mm Hg, indicating a graded relationship of NO production, increasing PaCO2, and increasing CBF. Diminution of CBF with NOSI infusion was reversed by simultaneous ICA infusion of l-arginine, indicating a direct role of NO synthesis in the chemoregulation of CBF. Hypotension and hypertension were induced with trimethaphan camsylate (Arfonad) and phenylephrine at constant PaCO2 (40 ± 1 mm Hg). Autoregulation in response to changes in MAP from 50 to 140 mm Hg was unaffected by ICA infusion of NOSI. In primates, cerebral vascular tone is modulated in vivo by NO; continuous release of NO is necessary to maintain homeostatic cerebral vasodilation; vasodilation during chemoregulation of CBF is mediated directly by NO production; autoregulatory vasodilation with hypotension is not mediated by NO; and increasing PaCO2 induces increased NO production.

scholarly journals Novel role of NF-κB-p65 in antioxidant homeostasis in human kidney-2 cells

2012 ◽  
Vol 302 (11) ◽  
pp. F1440-F1446 ◽  
Author(s):  
Liza E. George ◽  
Mustafa F. Lokhandwala ◽  
Mohammad Asghar
Keyword(s):  
Antioxidant Enzymes ◽  
Human Kidney ◽  
Redox Balance ◽  
Nuclear Factor Κb ◽  
D1 Receptor ◽  
Luciferase Reporter ◽  
Renal Cells ◽  
Direct Role ◽  
In Cells

Nuclear factor-κB (NF-κB) plays a role in inflammation. However, we recently reported an association between NF-κB and antioxidant enzymes in renal proximal tubules of exercise-trained rats, suggesting its role in antioxidant homeostasis (George L, Lokhandwala MF, Asghar M. Am J Physiol Renal Physiol 297: F1174–F1180, 2009). A direct role of NF-κB in antioxidant homeostasis in renal cells has not been elucidated and warrants investigation. Therefore, we examined whether NF-κB has a direct role in antioxidant homeostasis and redox balance in human kidney-2 cells overexpressing NF-κB-p65 and compared them with the cells overexpressing Nrf-2, a well-known transcription factor involved in antioxidant homeostasis. The ability of NF-κB-p65 to increase antioxidant enzymes, to reduce reactive oxygen species (ROS), and to rescue ROS-induced renal dopamine D1 receptor dysfunction, was studied. The transcription activity of NF-κB-p65 and Nrf-2, measured as luciferase reporter activity, increased in cells overexpressing these nuclear factors. The levels of mRNA and activity of glutathione peroxidase as well as the protein levels of superoxide dismutase-1 and glutamylcystein transferase were increased in cells overexpressing NF-κB-p65 and Nrf-2. Furthermore, the levels of ROS decreased and D1 receptor agonist SKF38393-mediated [35S]GTPγS binding (index of D1 receptor function) increased in the presence of hydrogen peroxide in cells overexpressing NF-κB-p65 and Nrf-2. These results suggest a direct role of NF-κB-p65 in antioxidant homeostasis, contributing to redox balance in renal cells.

scholarly journals Assessing the Mechanisms Responsible for Differences between Nitrogen Requirements of Saccharomyces cerevisiae Wine Yeasts in Alcoholic Fermentation

2013 ◽  
Vol 80 (4) ◽  
pp. 1330-1339 ◽  
Author(s):  
Claire Brice ◽  
Isabelle Sanchez ◽  
Catherine Tesnière ◽  
Bruno Blondin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcoholic Fermentation ◽  
Expression Patterns ◽  
Nitrogen Deficiency ◽  
Glycolytic Flux ◽  
Wine Yeasts ◽  
Content Type ◽  
Stress Genes ◽  
Essential Nutrient

ABSTRACTNitrogen is an essential nutrient forSaccharomyces cerevisiaewine yeasts during alcoholic fermentation, and its abundance determines the fermentation rate and duration. The capacity to ferment under conditions of nitrogen deficiency differs between yeasts. A characterization of the nitrogen requirements of a set of 23 strains revealed large differences in their fermentative performances under nitrogen deficiency, and these differences reflect the nitrogen requirements of the strains. We selected and compared two groups of strains, one with low nitrogen requirements (LNRs) and the other with high nitrogen requirements (HNRs). A comparison of various physiological traits indicated that the differences are not related to the ability to store nitrogen or the protein content. No differences in protein synthesis activity were detected between strains with different nitrogen requirements. Transcriptomic analysis revealed expression patterns specific to each of the two groups of strains, with an overexpression of stress genes in HNR strains and a stronger expression of biosynthetic genes in LNR strains. Our data suggest that differences in glycolytic flux may originate from variations in nitrogen sensing and signaling under conditions of starvation.

scholarly journals What next for end-point assessments?

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Denise Baker ◽  
David Robertshaw
Keyword(s):  
Design Methodology ◽  
Regulatory Compliance ◽  
Future Change ◽  
Direct Role ◽  
Content Type ◽  
End Point ◽  
Education And Health ◽  
Salient Points ◽  
Assessment Strategies

PurposeThis paper reflects on changes to end-point assessment (EPA) brought about as a result of the COVID-19 pandemic and considers how proposed future change will impact on training providers and employers of health apprentices.Design/methodology/approachThe paper provides an analysis of apprenticeship policy, the role of EPA and consideration of assessment strategies used in higher education and health professions. Implications for policy, training providers and clinical practice are proposed.FindingsThese changes will bring the completion of EPA closer to education providers and allow them to take a more direct role within the process. Education providers will need to be issued with clear guidance to ensure regulatory compliance. The pedagogical value of EPA is questioned.Originality/valueTraining providers and policymakers will need to review their processes and guidance appropriately. This paper provides a summary of salient points needing consideration.

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