oxidation

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

Redoxoma Highlights by Paolo Di Mascio
Corresponding author e-mail: pdm-I-am-here-ascio@hotmail.com@iq.usp.br

Singlet oxygen (¹O₂) is a biologically relevant reactive oxygen species capable of efficiently reacting with cellular constituents. Although it has usually received less attention than other free radical or non free-radical oxidants, the main oxidation reactions initiated by ¹O₂ and the resulting modifications within key cellular targets, including guanine for nucleic acids, unsaturated lipids, and targeted amino acids are increasingly evident and have been described in a recent publication [1]. Most of these reactions give rise to peroxides and dioxetanes, whose formation has been rationalized in terms of [4+2] cycloaddition and 1,2-cycloaddition with dienes + olefins, respectively [1].

Ultraweak chemiluminescence arising from biomolecules oxidation has been attributed to the radioative deactivation of ¹O₂ and electronically excited triplet carbonyl products involving dioxetane intermediates. As examples, the generation of ¹O₂ from lipid hydroperoxides, which involves a cyclic mechanism from a linear tetraoxide intermediate. Also, the generation of ¹O₂ via energy transfer from the excited triplet acetone arising from the thermodecomposition of dioxetane is a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal is an enzymatic source [2].

Recently Stanley and colleagues described a possible pathophysiological role for ¹O₂ in mammals, through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions [3,4].

Chemically generated ¹O₂ oxidizes the amino acid tryptophan (W) to precursors of a key metabolite called N-formylkynurenine (NFK), while enzymatic oxidation of W to NFK is catalyzed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1 (IDO1). Inflammation is associated with increased H₂O₂ and IDO1 also possesses peroxidase activity. Tryptophan oxidation by IDO1 in the presence of H₂O₂ revealed that cis-WOOH (a tricyclic triptophan-derived cis-hydroperoxide) is formed as the major product of a previously unrecognized oxidatively activated dioxygenase activity of IDO1. cis-WOOH is a precursor of NFK and the thermal decomposition of cis-WOOH also led to emission of light, characteristic of activated carbonyls [3–5]. The cis-WOOH acts as a hitherto undiscovered signaling molecule in vivo, which induces arterial relaxation and decreases blood pressure dependent on cysteine residue 42 of protein kinase (PK) G1. The IDO1/PKG1 axis can be a possible therapeutic target in sepsis, where oxidative activation of PKG1 and IDO1 activity are prevalent. Another possibility is that the reactive and potentially cytotoxic ¹O₂ may contribute to IDO1-mediated immune tolerance and tumor evasion, with inhibition of IDO1 representing a major target of anti-cancer drug development.

The approach used to unequivocally demonstrate the generation of ¹O₂ in these reactions is the use of ¹⁸O-labeled peroxides / triplet dioxygen (¹⁸[³O₂]), the detection of labeled compounds by HPLC coupled to mass spectrometry (HPLC-MSn) and the direct spectroscopic detection and characterization of ¹O₂ light emission. Characteristic light emission at 1,270 nm, corresponding to the singlet delta state monomolecular decay was observed. Using ¹⁸[³O₂], we observed the formation of ¹⁸O-labeled ¹O₂ (¹⁸[¹O₂]) by the chemical trapping of ¹⁸[¹O₂] with the anthracene-9,10-diyldiethane-2,1-diyl disulfate disodium salt (EAS) and detected the corresponding ¹⁸O-labeled EAS endoperoxide using HPLC-MS/MS [1].

The reactivity of ¹O₂ with biomolecules, as amino acids or lipids, may generate specific stereoselective oxidation products [1]. The elucidation of these structures and their specific targets can give important information and new horizons on the (patho)-physiological function for ¹O₂ in mammals via formation of signaling molecules.

References

1. P. Di Mascio, G. R. Martinez, S. Miyamoto, G. E. Ronsein, M. H. G. Medeiros, J. Cadet. Singlet Molecular Oxygen Reactions with Nucleic Acids, Lipids, and Proteins Chemical Reviews, 119(3): 2043–86, 2019. | doi: 10.1021/acs.chemrev.8b00554
2. C. M. Mano, F. M. Prado, J. Massari, G. E. Ronsein, G. R. Martinez, S. Miyamoto, J. Cadet, H. Sies, M. H. G. Medeiros, E. J. H. Bechara, et al.. Excited singlet molecular O₂ (¹Δ_g) is generated enzymatically from excited carbonyls in the dark Scientific Reports, 4(1): 2014. | doi: 10.1038/srep05938
3. G. E. Ronsein, M. C. B. Oliveira, S. Miyamoto, M. H. G. Medeiros, P. Di Mascio. Tryptophan Oxidation by Singlet Molecular Oxygen [O₂(¹Δ_g)]: Mechanistic Studies Using¹⁸O-Labeled Hydroperoxides, Mass Spectrometry, and Light Emission Measurements Chemical Research in Toxicology, 21(6): 1271–83, 2008. | doi: 10.1021/tx800026g
4. C. P. Stanley, G. J. Maghzal, A. Ayer, J. Talib, A. M. Giltrap, S. Shengule, K. Wolhuter, Y. Wang, P. Chadha, C. Suarna, et al.. Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation Nature, 566(7745): 548–52, 2019. | doi: 10.1038/s41586-019-0947-3
5. D. A. Kass. Fresh evidence overturns the identification of a factor involved in blood-vessel dilation Nature, 566(7745): 462–4, 2019. | doi: 10.1038/d41586-019-00508-z

Paolo Di Mascio, Full Professor at Department of Biochemistry,
Institute of Chemistry, University of São Paulo, Brazil