Redoxoma

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Singlet oxygen regulates vascular tone and blood pressure in inflammation

Researchers from the RIDC Redoxoma participated in the study published in the journal Nature
PorBy Maria Celia Wider
• CEPIDRIDC Redoxoma
08/03/2019
São Paulo, Braszil
Paolo Di Mascio
Paolo Di Mascio: personal collection

A study conducted by an international team of scientists, including the biochemists Paolo Di Mascio and Fernanda M. Prado, at Departamento de Bioquímica of the Instituto de Química of the Universidade de São Paulo (USP) and a members of the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma), showed for the first time that singlet oxygen plays a pathophysiological role in mammals through the formation of a signaling molecule that regulates vascular tone and blood pressure during acute systemic inflammation. The results of the study were published in the journal Nature and received a commentary in the News & Views section.

"Singlet oxygen is involved in the physiology of plants, bacteria and fungi. In mammals, it was seen as a species produced by cells as neutrophils to combat microorganisms or in photosensitization reactions. We now show that it forms a hydroperoxide derived from the amino acid tryptophan, which acts as a redox signaling molecule involved in the regulation of blood pressure in inflammation," Di Mascio said.

In pathological situations such as sepsis, there is an increase in the synthesis of nitric oxide by nitric oxide synthases, which leads the patient to hypotension and even death. Paradoxically, inhibitors of the nitric oxide pathway generally do not alleviate severe septic shock, suggesting the involvement of additional hypotension mediators, such as the one proposed in this paper. The results obtained may point to a new therapeutic target in sepsis.

The study, led by researcher Roland Stocker, at the Victor Chang Cardiac Research Institute in Australia, brought together researchers from Australia, England, Brazil, China, Germany, and Japan. RIDC Redoxoma is a Research, Innovation and Dissemination Center funded by FAPESP.

Tryptophan-derived hydroperoxide

Singlet oxygen is an electronically excited species of molecular oxygen. Although it exhibits high reactivity, oxidizing biological targets such as unsaturated fatty acids, proteins, and DNA, its role in biological systems is not yet well known.

Tryptophan is an essential amino acid that, besides being part of proteins, is a precursor of bioactive compounds. The chemically generated singlet oxygen oxidizes the amino acid tryptophan to precursors N-formylkynurenine (NFK) and Kynurenine (Kyn). The enzymatic oxidation of tryptophan to NFK is catalyzed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1 (IDO1). Under inflammatory conditions, this heme enzyme is expressed in arterial endothelial cells.

Singlet oxygen regulates vascular tone and blood pressure in inflammation

In 2010, the team of Stocker had published an article proposing that kynurenine was involved in vessel relaxation but, failing to repeat the results with posteriorly purchased kynurenine, concluded that the "relaxation factor" derived from tryptophan would probably be a metabolite ‘upstream’ of NFK. As products of chemical oxidation of tryptophan by oxygen singlet decay to NFK, commercial kynurenine could be contaminated by possible precursors. The researchers, then, exposed tryptophan to singlet oxygen in vitro and investigated all the products generated.

Eight different compounds were found and the researchers verified that only one of them substantially and dose-dependently relaxed arteries from rat, mouse and pig.

The compound was chemically confirmed as a tricyclic cis-hydroperoxide derived from tryptophan, cis-WOOH, by Di Mascio, who has extensive experience in the study of oxidations mediated by singlet oxygen. "In fact, this study began about ten years ago, when the then Ph.D. student in my lab, now a professor at IQ-USP and member of Redoxoma, Graziella E. Ronsein, determined the products of the reaction of singlet oxygen with the amino acid tryptophan."

Paolo Di MascioSeveral techniques have been used to study the compounds and identify the hydroperoxide, such as liquid chromatography coupled with mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR).

Di Mascio explains that in inflammation occurs a burst in hydrogen peroxide (H2O2) production, and the maintenance of these high stationary concentrations associated to the increase of IDO1 enzyme expression in an inflammatory situation leads to a gain of function of this enzyme. It goes from oxidase to ‘peroxidase’ and generates cis-WOOH hydroperoxide via singlet oxygen. The cis refers to the spatial configuration of the hydroperoxide. The enzymatic activity of IDO1 favors the formation of cis hydroperoxide in relation to trans, which already suggested that singlet oxygen is an actor formed in or near the active site of the enzyme.

The researchers found that this tryptophan hydroperoxide acts as a signaling molecule, inducing relaxation of arteries from different species and decreasing blood pressure in mice. This activity, according to them, depends on a specific cysteine ​​residue, cysteine-42, of protein kinase G1 α (PKG1α). Protein kinases play a key role in the regulation of intracellular signal transduction pathways.

With experiments performed on porcine coronary arteries treated with interferon-γ to increase IDO1 expression and mimic inflammatory conditions, the researchers proved that cis-WOOH is formed in vivo.

The authors point out that the findings suggest new biological functions for the IDO1 enzyme and may serve as a starting point for the discovery of a refined repertoire of redox signaling pathways. In addition to IDO1 being able to become a therapeutic target in sepsis, research data also indicate the possibility that singlet oxygen may be involved in the modulation of the immune response against tumors, via IDO1-mediated immune tolerance and tumor evasion, with inhibition of IDO1 representing a major target for drug development.

In addition, the study shifts focus from hydrogen peroxide to an amino acid derived hydroperoxide as a stereospecific signaling molecule. Other heme proteins known to generate singlet oxygen in the presence of hydrogen peroxide could also form products that, like cis-WOOH, act as signaling molecules.

The article “Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation”, by Christopher P. Stanley, Ghassan J. Maghzal, Anita Ayer, Jihan Talib, Andrew M. Giltrap, Sudhir Shengule, Kathryn Wolhuter, Yutang Wang, Preet Chadha, Cacang Suarna, Oleksandra Prysyazhna, Jenna Scotcher, Louise L. Dunn, Fernanda M. Prado, Nghi Nguyen, Jephthah O. Odiba, Jonathan B. Baell, Johannes-Peter Stasch, Yorihiro Yamamoto, Paolo Di Mascio, Philip Eaton, Richard J. Payne and Roland Stocker, can be accessed at https://www.nature.com/articles/s41586-019-0947-3

The commentary “Fresh evidence overturns the identification of a factor involved in blood-vessel dilation” can be accessed at https://www.nature.com/articles/d41586-019-00508-z