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Peroxiredoxin hyperoxidation increases in the presence of bicarbonate/carbon dioxide

Submitted by redoxoma on
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 CPSOH to CPSO2– 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

  1. 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
  2. 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
  3. 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
  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
  5. 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
  6. 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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Revealing the cross-talk between nitric oxide metabolites

Submitted by redoxoma on
Artistic EPR spectra

Redoxoma Highlights, by Daniela Truzzi
Corresponding author e-mail: dtruz@hotmail.comzi@iq.usp.br

Nitric oxide (NO•) is an endogenously produced diatomic radical that regulates fundamental biological functions. Although NO• is a free radical, its reactivity in biological media is selective toward other radicals and transition metal centers. NO• metabolites include S-nitroso thiols (RSNOs), nitrite, peroxynitrite, nitrosylated heme proteins, and dinitrosyl-iron complexes (DNICs). Among these metabolites, RSNOs have gained considerable attention due to their possible involvement in NO• signaling. Biological formation of RSNO can occur by reaction of thiols with N2O3, peroxynitrite, other S-nitroso thiols (transnitrosation reactions), nitrosylated heme proteins and by a direct reaction between thiyl radicals (RS•) and NO•. Since most of these reactions are either slow or have low specificity for a signaling process, it has been proposed that S-nitrosation involves transfer of NO from DNICs to biothiols. DNICs are important NO•-metabolites, which are able to trigger vasodilation, to inhibit platelet aggregation and skin wound healing. Nevertheless, little is known about the dynamics of DNICs generation under physiological conditions. By analyzing DNIC assemble from the reaction between NO•, Fe(II) and low molecular weight biothiols (cysteine and glutathione) in aqueous media, pH 7.4, we detected mono-nitrosyl iron complex intermediate(s) and thiyl radicals (RS•) as co-products. By demonstrating that formation of DNICs yields RS• in a NO• rich environment, these results provide a novel route for S-nitroso thiol formation in biological media. Additionally, this study explains previous reports showing that DNICs and RSNOs are simultaneously formed in macrophages exposed to NO•. If such mechanism favors certain biothiols in forming RS• and thus provides specificity to RSNO formation, remains an open question. Further studies of DNICs assembly with different biothiols may contribute to answer it.

Importantly, different biothiols may react through distinct mechanism with DNICs, opening the question if all biothiols would similarly have RS• as an intermediate. This is of high relevance when considering signaling pathways and specificity.

This study involved collaboration between researchers from CEPID Redoxoma and from University of California, Santa Barbara.

Related article:

  1. D. R. Truzzi, O. Augusto, P. C. Ford. Thiyl radicals are co-products of dinitrosyl iron complex (DNIC) formation Chemical Communications, 55(62): 9156–9, 2019. | doi: 10.1039/c9cc04454j

Daniela Truzzi, from Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil

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A link between mitochondrial shape and function in Ca²⁺ signaling

Submitted by redoxoma on
Calcium

Redoxoma Highlights by Sergio Menezes and Alicia Kowaltowski
Corresponding author e-mail: alicia@kowaltowski@iq.usp.br

Differently from what one might expect from the textbook representations, mitochondria are not static organelles which are always in well-defined round shapes. In fact, they are highly dynamic organelles which undergo constant cycles of fission and fusion with nearby mitochondria [1]. “mitochondria are not static organelles … in well-defined round shapes” The balance between these fusion and fission events within the cell is what will define the shape of the mitochondrial network, which can range all the way from several separate round shaped mitochondria (high fission, low fusion balance) to very interconnected networks of elongated mitochondria that span throughout the cell (high fusion, low fission balance) [1]. Several factors like cell type, nutrient availability, progression through the cell cycle and even pathological conditions contribute to this fusion/fission balance within the cell [2, 3]. The study of these dynamic modulations of mitochondrial morphology is broadly called "mitochondrial dynamics" [1], and is a field that has received a lot of attention in the last two decades.

In our recent study accepted for publication in The Faseb Journal [preprint available at https://www.biorxiv.org/content/10.1101/624981v1], we show a novel role for mitochondrial dynamics in regulating cellular Ca2+ signaling and homeostasis. “we show a novel role for mitochondrial dynamics in regulating cellular Ca2+ signaling and homeostasis” Mitochondria forced into a pro-fusion phenotype through the inhibition of the fission protein DRP1 (by competition with a dominant-isoform) presented increased mitochondrial Ca2+ uptake rates and maximal uptake capacity, while mitochondria forced to a fission phenotype by the knockdown of the fusion protein MFN2 showed the opposite effect. Mitochondrial Ca2+ uptake is a process mediated by the entry of Ca2+ ions in the mitochondrial matrix through the protein MCU (mitochondrial calcium uniporter) driven by the negative-inside mitochondrial membrane potential, and has been extensively shown to impact several processes involving Ca2+ signaling in the cell [6]. In our work we also show that one of these regulated Ca2+ uptake processes, known as store-operated Ca2+ entry (a homeostatic mechanism by which cells are capable of replenishing their ER Ca2+ stores by promoting extracellular Ca2+ entry through the membrane) is modulated by mitochondrial morphology, with more fragmented mitochondria resulting in an impairment of this process, while more fused mitochondria resulted in a faster activation of extracellular Ca2+ entry. We also have observed a reduction in basal cytoplasmic and ER Ca2+ levels in the cells with more fragmented mitochondria, which was associated with increased levels of ER stress markers, showing that mitochondrial morphology can also regulate these aspects of cellular Ca2+ homeostasis.

By showing that the modulation of mitochondrial morphology can impact mitochondrial Ca2+ uptake and promote changes in Ca2+ homeostasis in the cell, this work establishes a new connection between mitochondrial dynamics and cell signaling.

References

  1. L. Tilokani, S. Nagashima, V. Paupe, J. Prudent. Mitochondrial dynamics: overview of molecular mechanisms Essays In Biochemistry, 62(3): 341–60, 2018. | doi: 10.1042/ebc20170104
  2. M. Liesa, O. Shirihai. Mitochondrial Dynamics in the Regulation of Nutrient Utilization and Energy Expenditure Cell Metabolism, 17(4): 491–506, 2013. | doi: 10.1016/j.cmet.2013.03.002
  3. R. Horbay, R. Bilyy. Mitochondrial dynamics during cell cycling Apoptosis, 21(12): 1327–35, 2016. | doi: 10.1007/s10495-016-1295-5
  4. M. F. Forni, J. Peloggia, K. Trudeau, O. Shirihai, A. J. Kowaltowski. Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics Stem Cells, 34(3): 743–55, 2015. | doi: 10.1002/stem.2248
  5. M. Vig, J. Kinet. Calcium signaling in immune cells Nature Immunology, 10(1): 21–7, 2008. | doi: 10.1038/ni.f.220
  6. A. Spät, G. Szanda, G. Csordas, G. Hajnóczky. High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling Cell Calcium, 44(1): 51–63, 2008. | doi: 10.1016/j.ceca.2007.11.015

Sergio Menezes and Alicia Kowaltowski, from Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil

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Neutrophils add another layer of complexity to redox signaling

Submitted by redoxoma on
neutrophil

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

  1. 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
  2. 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
  3. 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
  4. 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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