Article

A Model of Redox Kinetics Implicates the Thiol Proteome in Cellular Hydrogen Peroxide Responses

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
Antioxidants & Redox Signaling (Impact Factor: 7.41). 09/2010; 13(6):731-43. DOI: 10.1089/ars.2009.2968
Source: PubMed

ABSTRACT

Hydrogen peroxide is appreciated as a cellular signaling molecule with second-messenger properties, yet the mechanisms by which the cell protects against intracellular H(2)O(2) accumulation are not fully understood. We introduce a network model of H(2)O(2) clearance that includes the pseudo-enzymatic oxidative turnover of protein thiols, the enzymatic actions of catalase, glutathione peroxidase, peroxiredoxin, and glutaredoxin, and the redox reactions of thioredoxin and glutathione. Simulations reproduced experimental observations of the rapid and transient oxidation of glutathione and the rapid, sustained oxidation of thioredoxin on exposure to extracellular H(2)O(2). The model correctly predicted early oxidation profiles for the glutathione and thioredoxin redox couples across a range of initial extracellular [H(2)O(2)] and highlights the importance of cytoplasmic membrane permeability to the cellular defense against exogenous sources of H(2)O(2). The protein oxidation profile predicted by the model suggests that approximately 10% of intracellular protein thiols react with hydrogen peroxide at substantial rates, with a majority of these proteins forming protein disulfides as opposed to protein S-glutathionylated adducts. A steady-state flux analysis predicted an unequal distribution of the intracellular anti-oxidative burden between thioredoxin-dependent and glutathione-dependent antioxidant pathways, with the former contributing the majority of the cellular antioxidant defense due to peroxiredoxins and protein disulfides.

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    • "Extra intracellular H 2 O 2 degrades through the cascade of oxidation/reduction reactions of the enzymes involving in ROS cellular scavenging systems (module 9, Fig. 1). The model reproduced the typical kinetics of peroxidase oxidised during treatment cells by H 2 O 2 (Adimora et al., 2010) (blue line, Fig. 9). The total concentration of peroxidase (Px) in the model increased, and this indicates a NRF2-KEAP1-dependent expression of peroxidase in response to redox perturbation (red line, Fig. 9). "
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    ABSTRACT: Cells are constantly exposed to Reactive Oxygen Species (ROS) produced both endogenously to meet phys- iological requirements and from exogenous sources. While endogenous ROS are considered as important signalling molecules, high uncontrollable ROS are detrimental. It is unclear how cells can achieve a bal- ance between maintaining physiological redox homeostasis and robustly activate the antioxidant system to remove exogenous ROS. We have utilised a Systems Biology approach to understand how this robust adaptive system fulfils homeostatic requirements of maintaining steady-state ROS and growth rate, while undergoing rapid readjustment under challenged conditions. Using a panel of human ovarian and normal cell lines, we experimentally quantified and established interrelationships between key elements of ROS homeostasis. The basal levels of NRF2 and KEAP1 were cell line specific and maintained in tight corre- lation with their growth rates and ROS. Furthermore, perturbation of this balance triggered cell specific kinetics of NRF2 nuclear–cytoplasmic relocalisation and sequestration of exogenous ROS. Our experi- mental data were employed to parameterise a mathematical model of the NRF2 pathway that elucidated key response mechanisms of redox regulation and showed that the dynamics of NRF2-H2O2 regulation defines a relationship between half-life, total and nuclear NRF2 level and endogenous H2O2 that is cell line specific.
    Full-text · Article · Nov 2014 · Journal of Biotechnology
    • "Although structural and kinetic information are available on the catalytic mechanisms of these enzymes (TrxR and Prx) [17– 24,28–32], the mathematical models developed so far to represent their kinetics are not well elucidated and are restricted to simple mass action kinetics [33] [34]. Furthermore, none of the developed models are able to describe the experimentally observed NADPH-mediated substrate inhibition of TrxR and pHmediated bell-shaped behavior of the enzyme activity. "
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    No preview · Article · Oct 2014 · Free Radical Biology and Medicine
    • "Nevertheless, important aspects of this system and how its design relates to function remain unclear. Mathematical modeling has consistently proved useful in clarifying the mechanisms of antioxidant defense and redox signaling [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]. Kinetic models help identify gaps and inconsistencies in the state of the art, assessing alternative mechanistic hypotheses, understanding the interplay among multiple factors, and understanding the relationship between molecular-level design and phenotype. "
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    ABSTRACT: H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2’s high reactivity with H2O2 (kPrx2=10×107 M-1s-1, kCat =7×107 M-1s-1, kGPx1 =4×107 M-1s-1) and high abundance ([Prx2]= 570 µM, [Cat]= 32 µM, [GPx1]= 1 µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2’s effective rate constant with H2O2would have to be just ~105 M-1s-1, much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1 min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=108 M-1s-1 shows important qualitative discrepancies. Altogether, these results suggest that Prx2’s peroxidase activity is strongly inhibited in human erythrocytes.
    No preview · Article · Oct 2014
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