peroxiredoxin

Peroxiredoxin hyperoxidation increases in the presence of bicarbonate/carbon dioxide

Submitted by redoxoma on Fri, 02/28/2020 - 18:34

peroxidase cycle, hyperoxidation pathway and interaction and oxidation of other thiol proteins, signaling

Highlights by Daniela R. Truzzi and Ohara Augusto, from Instituto de Química da USP
Corresponding author e-mail: dtruz-I-am-here-zi@hotmail.com@iq.usp.br

Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H₂O₂, constituting an important antioxidant defense and acting as sensors and transmitters of H₂O₂ signals in cells. Eukariotic 2-Cys Prxs lose their peroxidase activity at high hydroperoxide levels in a process called hyperoxidation, which constitutes a pathway to Prx functions beyond the antioxidant activity (Fig. 1) [1]. Since the biologically ubiquitous HCO₃^–/CO₂ pair accelerates the reaction of H₂O₂ with several biothiols [2, 3], we examined whether physiological concentrations of the HCO₃^–/CO₂ pair (25 mM) could increase recombinant human peroxiredoxin 1 (Prx1) hyperoxidation by H₂O₂ [4]. Immunoblotting, kinetic and MS/MS experiments revealed that HCO₃^–/CO₂ increases Prx1 hyperoxidation and inactivation both in the presence of excess H₂O₂ and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (dithiothreitol) turnover. Based on previous studies, we hypothesized that the stimulating effect of HCO₃^–/CO₂ was due to HCO₄^– (peroxymonocarbonate), a peroxide present in equilibrated solutions of H₂O₂ and HCO₃^–/CO₂ [2, 3]. Indeed, additional experiments and calculations uncovered that HCO₄^– oxidizes C_PSOH to C_PSO₂^– with a second-order rate constant two orders of magnitude higher than H₂O₂ ((1.5 ± 0.1) × 10⁵ and (2.9 ± 0.2) × 10³ M⁻¹.s⁻¹, respectively) and that HCO₄^– is 250 times more efficient than H₂O₂ at inactivating 1% Prx1 per turnover [4]. The fact that the biologically ubiquitous HCO₃^–/CO₂ pair stimulates Prx1 hyperoxidation may be quite relevant to cell homeostasis because the antioxidant and redox relay functions of the enzyme decline but other actions may rise, such as the chaperone-like activity, the redox signaling pathways mediated by Cys-based proteins that are poorly reactive towards H₂O₂ and the maintenance of Trx-dependent activities (Fig. 1). Relevantly, parallel studies led by Christine Winterbourn and co-workers reported that the HCO₃^–/CO₂ pair also stimulates Prx2 and Prx3 hyperoxidation [5] and protein tyrosine phosphatase 1B inactivation involved in epidermal growth factormediated signaling [6]. Taking together, these recent studies confirm that HCO₄^– deserves further investigation as a biological oxidant [3] and point to a possible role of HCO₃^–/CO₂ levels in H₂O₂mediated signaling.

Peroxidase cycle, hyperoxidation pathway

Figure 1. Simplified scheme of the catalytic cycle and the hyperoxidation pathway of 2-Cys Prxs and the activities related to them.

References

E. A. Veal, Z. E. Underwood, L. E. Tomalin, B. A. Morgan, C. S. Pillay. Hyperoxidation of Peroxiredoxins: Gain or Loss of Function? Antioxidants & Redox Signaling, 28(7): 574–90, 2018. | doi: 10.1089/ars.2017.7214
D. F. Trindade, G. Cerchiaro, O. Augusto. A Role for Peroxymonocarbonate in the Stimulation of Biothiol Peroxidation by the Bicarbonate/Carbon Dioxide Pair Chemical Research in Toxicology, 19(11): 1475–82, 2006. | doi: 10.1021/tx060146x
D. R. Truzzi, O. Augusto. Influence of CO₂ on Hydroperoxide Metabolism Hydrogen Peroxide Metabolism in Health and Disease, (Vissers, M.C.M., Hampton, M., Kettle, A.J. eds) 81–99, Oxidative stress and disease, Taylor & Francis/CRC Press, Boca Raton, 2017. | doi: 10.1201/9781315154831-4
D. R. Truzzi, F. R. Coelho, V. Paviani, S. V. Alves, L. E. S. Netto, O. Augusto. The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1 Journal of Biological Chemistry, 294(38): 14055–67, 2019. | doi: 10.1074/jbc.ra119.008825
A. V. Peskin, P. E. Pace, C. C. Winterbourn. Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO₂ Free Radical Biology and Medicine, 145: 1–7, 2019. | doi: 10.1016/j.freeradbiomed.2019.09.010
M. Dagnell, Q. Cheng, S. H. M. Rizvi, P. E. Pace, B. Boivin, C. C. Winterbourn, E. S. J. Arnér. Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades Journal of Biological Chemistry, 294(33): 12330–8, 2019. | doi: 10.1074/jbc.ra119.009001

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Neutrophils add another layer of complexity to redox signaling

Submitted by redoxoma on Wed, 05/15/2019 - 18:52

Redoxoma Highlights by Luiz Felipe de Souza

Neutrophils kill invading microbes with a myriad of antimicrobial agents and powerful oxidants. Phagocytosis and other stimuli activate the NADPH oxidase complex that generates high amounts of oxidants, which are crucial to host defense but can also promote damage to host tissue and jeopardize neutrophil function. neutrophils were traditionally considered to be a “kamikaze” cell Thus, neutrophils were traditionally considered to be a “kamikaze” cell, dying quickly after activation due to self-inflicted oxidative damage. However, it is becoming clear that neutrophils can survive for several hours after activation and are important both for regulation and termination of inflammation by releasing cytokines and other inflammatory mediators. This suggests that neutrophils must have highly efficient antioxidant systems to protect ongoing functions. We examined this hypothesis by studying peroxiredoxin in these cells [1].

Peroxiredoxins (Prxs) are thiol peroxidases that are particularly sensitive to oxidation. Prxs rely on highly reactive cysteine thiols for their antioxidant functions. Because of their high abundance and fast reactivity, Prxs are thought to act as peroxide sensors, normally being in their reduced form, and becoming oxidized as the environment shifts towards a more oxidizing state [2]. This holds true for most mammalian cells, but this system seems to be more complicated in neutrophils. By analyzing the redox state of the cytoplasmic Prxs (Prx1 and 2) in non-stimulated neutrophils, we detected that almost all the Prx pool was already oxidized in these conditions [1]. Prxs of other lymphocytes taken from the same blood showed only about 15-20% of Prx oxidation, indicating that this phenomenon might be unique to neutrophils. Inhibition of the NAPDH oxidase complex or stimulation of oxidative burst did not change the oxidative state of Prx, suggesting that the recycling system may be deficient in neutrophils. Most puzzling, thioredoxin and thioredoxin reductase, the two enzymes directly responsible for Prx recycling, were expressed at reasonable levels in neutrophils, and in contrast to Prx, thioredoxin was mainly reduced in these cells. In contrast, Prxs in acute promyelocytic leukemia HL-60 cells induced to differentiate towards a neutrophil-like phenotype were reduced under non-stimulated conditions and became oxidized upon phagocytosis. Intriguingly, differentiation of the HL-60 cells by both retinoic acid and dimethyl sulfoxide let to a robust down-regulation of Prx1, indicating that this protein may play a role in the differentiation process [1].

These data raise a number of questions regarding the redox metabolism of neutrophils. It is known that oxidation of Prxs can control cell signaling by a disulfide relay mechanism. For example, Prx1 oxidation can activate apoptosis by promoting the oxidation of ASK1, and Prx2 can oxidize and repress STAT3 signaling [3,4]. So, how are these systems operating inside the neutrophils? And why does Prxs appear “locked” as disulfides when other thiols such as thioredoxin and glutathione are reduced? Perhaps trying to answer these questions may bring other exciting findings in the field of redox research.

References

L. F. de Souza, A. G. Pearson, P. E. Pace, A. L. Dafre, M. B. Hampton, F. C. Meotti, C. C. Winterbourn. Peroxiredoxin expression and redox status in neutrophils and HL-60 cells Free Radical Biology and Medicine, 135: 227–34, 2019. | doi: 10.1016/j.freeradbiomed.2019.03.007
R. A. Poynton, M. B. Hampton. Peroxiredoxins as biomarkers of oxidative stress Biochimica et Biophysica Acta (BBA) - General Subjects, 1840(2): 906–12, 2014. | doi: 10.1016/j.bbagen.2013.08.001
R. M. Jarvis, S. M. Hughes, E. C. Ledgerwood. Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells Free Radical Biology and Medicine, 53(7): 1522–30, 2012. | doi: 10.1016/j.freeradbiomed.2012.08.001
M. C. Sobotta, W. Liou, S. Stöcker, D. Talwar, M. Oehler, T. Ruppert, A. N. D. Scharf, T. P. Dick. Peroxiredoxin-2 and STAT3 form a redox relay for H₂O₂ signaling Nature Chemical Biology, 11(1): 64–70, 2014. | doi: 10.1038/nchembio.1695

Luiz Felipe de Souza, Pos-doc at Laboratory of Redox Processes in Inflammation at Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil

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Inflammatory oxidative burst: a new actor in this scenario

Submitted by redoxoma on Tue, 12/11/2018 - 11:51

Redoxoma Highlights by Flavia Carla Meotti

Activation of neutrophils either by invader microorganisms or by endogenous stimuli (sterile inflammation) triggers the assembling of NADPH oxidase at the membrane and the sequential production of free radicals and oxidants. This so-called inflammatory oxidative burst is initiated by the reduction of oxygen to superoxide, followed by the dismutation of superoxide to hydrogen peroxide. Hydrogen peroxide is a substrate to the heme-peroxidase, myeloperoxidase, oxidizing halides (mainly chloride) to hypohalous (hypochlorous) acid. Reaction between hydrogen peroxide and hypochlorous acid generates singlet oxygen. In parallel, the up-regulation of inducible nitric oxide synthase produces nitric oxide, which rapidly reacts with superoxide to form peroxynitrite. These are the main known players in the inflammatory oxidative burst. However, myeloperoxidase is a versatile enzyme and can effortlessly use hydrogen peroxide to oxidize other substrates in addition to halides. For instance, myeloperoxidase efficiently oxidizes uric acid to produce urate free radical, a short living reactive species that can rapidly combine with superoxide to generate an organic peroxide, urate hydroperoxide [1]. Although the formation of urate hydroperoxide was chemically feasible, the question was whether it would be biologically relevant since its precursor, urate free radical, could react with an unacountable number of biomolecules besides superoxide. This was one of the subjects addressed by our group at the Laboratory of Redox Process in Inflammation, CEPID-Redoxoma. By using mass spectrometry, we unambiguosly demonstrated the presence of urate hydroperoxide in stimulated peripheral blood neutrophils. Quantification of the amount of urate hydroperoxide that is formed by these inflammatory cells revealed that it is comparable to the amount of HOCl [2].

By analyzing the reactivity of urate hydroperoxide, we detected that it reacts preferentially with thiol groups in proteins and can rapidly oxidize the thiol peroxidases peroxiredoxin 1 (Prx1) and peroxiredoxin 2 (Prx2) [3]. These ubiquitous proteins are the fastest to react with hydrogen peroxide (~1 ⋅ 10⁸ M^-1s^-1) [4] and have been proposed as hydrogen peroxide sensors, able to perform a signaling redox relay mechanism [5]. Urate hydroperoxide oxidizes purified Prx1 and Prx2 at the rate constants of 4.5 ⋅ 10⁵ M^-1s^-1 and 2.3 ⋅ 10⁶ M^-1s^-1, respectively, and oxidized Prx2 from intact erythrocytes at the same extent as hydrogen peroxide [3]. Together, these data suggest urate hydroperoxide as a new reported protagonist in the inflammatory oxidative burst. The oxidation of cytosolic Prx1 and Prx2 by urate hydroperoxide might affect cell function and be partially responsible for the harmfull effects attibuted to uric acid.

References

F. C. Meotti, G. N. L. Jameson, R. Turner, D. T. Harwood, S. Stockwell, M. D. Rees, S. R. Thomas, A. J. Kettle. Urate as a Physiological Substrate for Myeloperoxidase Journal of Biological Chemistry, 286(15): 12901–11, 2011 | doi: 10.1074/jbc.m110.172460
R. P. Silva, L. A. Carvalho, E. S. Patricio, J. P. Bonifacio, A. B. Chaves-Filho, S. Miyamoto, F. C. Meotti. Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions Free Radical Biology and Medicine, 126: 177–86, 2018 | doi: 10.1016/j.freeradbiomed.2018.08.011
L. A. C. Carvalho, D. R. Truzzi, T. S. Fallani, S. V. Alves, J. C. Toledo, O. Augusto, L. E. S. Netto, F. C. Meotti. Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2 Journal of Biological Chemistry, 292(21): 8705–15, 2017 | doi: 10.1074/jbc.m116.767657
S. Portillo-Ledesma, L. M. Randall, D. Parsonage, J. Dalla Rizza, P. A. Karplus, L. B. Poole, A. Denicola, G. Ferrer-Sueta. Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing Biochemistry, 57(24): 3416–24, 2018 | doi: 10.1021/acs.biochem.8b00188
C. C. Winterbourn, M. B. Hampton. Signaling via a peroxiredoxin sensor Nature Chemical Biology, 11(1): 5–6, 2014 | doi: 10.1038/nchembio.1722

Flavia C. Meotti, Ph.D. Professor at Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil