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Redox Homeostasis and Signaling - University of Nebraska–Lincoln
Redox Homeostasis and Signaling Dmitri Fomenko University of Nebraska - Lincoln Percent of oxygen in earth atmosphere, % Evolution and oxygen 21% Oxygen 20 Start of shelled invertebrates 15 Start of multicelled plants 10 5 Start of photosynthesis Build up Ozone layer Start of respiration First living cell 0 4 3 2 1 Billion Years before Today Bacteria Mitochondria 0 Eukaryotes "LUCA" Archaea Redox homeostasis Redox homeostasis is the tendency of a cell to maintain internal ROS level by coordinated response to any situation or stimulus that would tend to disturb its normal condition. Redox processes requires the simultaneous presence of both oxidized and reduced forms of electron carriers. Reductants Oxidants Reductants Redox balance Oxidants Oxidative environment, Oxidative stress Oxidants Reductants Reductive environment, Reductive stress Controlled ROS generation Oxygen toxicity was mitigated during evolution by the development of oxidative stress defense systems and signaling systems for the control of intracellular ROS. The level of ROS is controlled not only by environmental and metabolic processes but also by ROS generation. ROS-based signaling that involves generation of ROS for signal transduction has evolved as a complex and essential process in eukaryotes. The NADPH oxidase (NOX) family of enzymes is important source of H2O2 for physiological redox signaling. A diverse array of extracellular signals can trigger the activation of NOX enzymes. H2O2 generated from active NOX targets redoxsensitive amino acids in signaling proteins (PTP1B, PTEN). Carroll, Nature Chemical Biology, 2011 ROS - the good and the bad sides - Gpx1 overexpression - hyperinsulinemia and insulin resistance - Gpx1 knockout - low level of insulin, high insulin sensitivity H2O2 Haque et al, Cell, 2011 ROS sources and defense systems Major intracellular ROS sources: Environmental factors influencing ROS level: - Mitochondria (respiration) - ER (oxidative protein folding) - NADPH oxidases - Reduced metals (Fenton's reaction) - Environmental oxidants (O2, H2O2) - Ionizing radiation Catalase O2 ē ._ ē,2H+ O2 H2O2 ē OH Superoxide dismutase _ . OH ē,H+ Peroxidases – PRXs, GPXs Thiol oxidoreductases H2O General functions of thiol oxidoreductases ROS detoxification Sulfur metabolism RNS detoxification Set of thiol oxidoreductases in an organism - THIOREDOXOME Transcription control Signal transduction Protein degradation Protein modification Oxidative folding 120 110 100 90 80 70 60 50 40 30 20 10 0 Thiol oxidoreductases Glycolytic enzymes 0 2000 4000 6000 Proteome size Number of identified proteins Number of identified proteins Identified thioredoxomes 8000 10000 1000 100 10 Eukaryota Bacteria Archaea 1 500 5000 50000 Proteome size Fomenko and Gladyshev, ARS, 2011 Thioredoxomes and environmental factors Number of identified proteins 100 80 60 40 Hyperthermophilic bacteria Series1 Thermophilic mez ps Mesophilic bacteria ter Psychrophilic bacteria 20 Number of identified proteins 120 120 100 0 80 60 40 20 0 0 2000 4000 6000 8000 10000 0 Proteome size 2000 4000 6000 8000 10000 Proteome size 120 120 100 80 60 40 20 Host associated bacteria Other bacteria 0 Number of identified proteins Number of identified proteins Series1 an Aerobic bacteria fa Microaerobic bacteria ma Facultative anaerobic bacteria Anaerobic bacteria 100 80 60 40 Host associated bacteria Terrestrial bacteria Specialized bacteria Aquatic bacteria 20 0 0 2000 4000 6000 Proteome size 8000 10000 0 2000 4000 6000 Proteome size 8000 10000 Thioredoxomes summary Minimal Thioredoxome – 3 thiol oxidoreductases Nanoarchaeum equitans 535 ORFs/3 thiol oxidoreductases – Peroxiredoxin, Thioredoxin and Thioredoxin reductase Largest Thioredoxome (194 proteins) - Arabidopsis thaliana Fomenko and Gladyshev, ARS, 2011 Redox stress response in unicellular and multicellular organisms In bacteria and unicellular eukaryotes, the induced expression of detoxifying enzymes in response to ROS plays a major role in protecting the cell against oxidative damage. In multicellular organisms, the increased expression of antioxidant enzymes is not a universal response of all cells to ROS, however, the basal levels of antioxidants is important for maintenance of homeostatic conditions and protection of cells against the damaging effects of oxidative stress. Such difference may relate to the high ROS concentrations that unicellular organisms can be exposed to within their ecological habitat, requiring appropriate fast response and adaptations. Human thioredoxome. 136 proteins, including 111 with catalytic Cys and 25 selenoproteins Fomenko and Gladyshev, ARS, 2011 Saccharomyces cerevisiae thioredoxome (47 proteins) Current model of H2O2 mediated signaling NADPH Thioredoxin reductase Thioredoxin Thioredoxin peroxidase Kinases Signaling Transcription factors Redox regulation Transcription response Other targets Current model H2O2 Targets H2O2 Molecular Mechanism for the Yap1-Gpx3 Redox System Paulsen CE et al, Chem. & Biol., 2009 Viability of WT cells and 8∆ cells Fraction viable Fraction Replicative life span Gpx1∆ ∆ Gpx2∆ ∆ Gpx3∆ ∆ 3Gpx∆ ∆ 5Gpx∆ ∆ 7∆ ∆ (all Prx∆ ∆ 2Gpx∆ ∆) 8∆ ∆ (all Prx∆ ∆ all Gpx∆ ∆) WT 1 0.8 0.6 0.4 0.2 0 0 10 20 30 Generations Generations 40 20 30 Generations Generations 40 WT Tsa1∆ Tsa1∆ Tsa2∆ Tsa2∆ Tsa1∆ Tsa1∆ Tsa2∆ Tsa2∆ nPrx∆ nPrx∆ mPrx∆ mPrx∆ Ahp1∆ Ahp1∆ Fraction viable Fraction viable 1 0.8 0.6 0.4 0.2 0 0 10 Regulation of gene expression in response to hydrogen peroxide 1. Deletion of single thiol peroxidase did not affect response to H2O2. 2. Yeast strains, which lack multiple thiol peroxidases are unable respond to H2O2 stress. 3. Minimal H2O2 stress response was observed in strain lacking all 8 thiol peroxidases. NADPH Thioredoxin reductase Signaling Redox regulation Transcription response Thioredoxin Thioredoxin peroxidase Kinases Transcription factors Other targets H2O2 ROS level in WT and 8∆ cells Catalase activity in WT and mutant strains. SOD activity in WT and mutant strains Exposed thiols and oxidized Cys in WT and mutant strains Glutathione content in WT and mutant strains 27 µM M / mg of total protein 28 5 7 21 17 25 21 23 21 21 24 8 5 27 19 17 32 27 23 15 20 8 32 16 22 Hydrogen peroxide response in thioredoxin mutant strains NADPH Thioredoxin reductase Thioredoxin peroxidase H2O2 Kinases Signaling Transcription factors Redox regulation Transcription response C Thioredoxin D Other targets E Regulation of gene expression by H2O2 and diamide WT activated by H2O2 (897) 787 WT repressed by H2O2 (923) 790 WT activated by diamide (1439) WT repressed by diamide (1313) WT activated by H2O2 (897) WT repressed by H2O2 (923) 756 8∆ ∆ activated by diamide (1498) 767 8∆ ∆ repressed by diamide (1337) W T Y ap Sk 1∆ n Y 7∆ ap M 1∆, s S 4∆ n2,4 kn ∆ 7∆ Regulation of gene expression in response to hydrogen peroxide in the mutants lacking redox transcription factors New model of H2O2-mediated signaling in S. cerevisiae Kinases NADPH Thioredoxin reductase Transcription factors Thioredoxins Thioredoxin peroxidases H2O2 Other targets Transcription response Redox regulation Current model H2O2 Targets Signaling New model H2O2 Thiol peroxidases Targets Model for PRDX4-Mediated Oxidative Protein Folding in the ER Disulfide bond formation Zito et al, Mol Cell, 2010 The role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway Jarvis et al, Free Radical Biology and Medicine, 2012
