Sensitive Detection and Estimation of Cell-Derived Peroxynitrite Fluxes Using Fluorescein-Boronate

Ríos, Natalia; Piacenza, Lucı́a; Trujillo, Madia; Martinez, Alejandra; Prolo, Carolina; Álvarez, María Noel; López, Gloria V.; Radí, Rafael

doi:10.1016/j.freeradbiomed.2016.10.073

Cited by 14 publications

(27 citation statements)

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“…The rate constant of the reaction, however, is significantly higher (~10 6 M -1 s -1 for ONOOand ~1 M -1 s -1 for H2O2), and the reaction typically involves a minor pathway, leading to ONOO -specific minor products (59). The high rate constant provides an opportunity to estimate the absolute flux of ONOOin cultured cells (48,60,61). Formation of ONOO --specific products provides an opportunity to selectively monitor ONOOformation in chemical and biological systems (14).…”

Section: Peroxynitritementioning

confidence: 99%

Tracking isotopically labeled oxidants using boronate-based redox probes

Ríos

Radí

Kalyanaraman

et al. 2020

Journal of Biological Chemistry

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Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18–labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

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Section: Peroxynitritementioning

confidence: 99%

Tracking isotopically labeled oxidants using boronate-based redox probes

Ríos

Radí

Kalyanaraman

et al. 2020

Journal of Biological Chemistry

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“…•− and • NO and thus peroxynitrite (58,59). First, SIN-1 decay (0.1 mM) was evaluated spectrophotometrically, identifying an isosbestic point at 250 nm (Fig.…”

Section: Fe-sodb Overexpression Lowers Peroxynitrite Generation Insidmentioning

confidence: 99%

Cytosolic Fe-superoxide dismutase safeguardsTrypanosoma cruzifrom macrophage-derived superoxide radical

Martínez

Prolo

Estrada

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During T. cruzi invasion to macrophages, superoxide radical (O2•−) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work, T. cruzi cytosolic Fe-SODB overexpressers (pRIBOTEX–Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox−/− macrophages, underscoring the role of O2•− in parasite killing. Herein, we studied the entrance of O2•− and its protonated form, perhydroxyl radical [(HO2•); pKa = 4.8], to T. cruzi at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2•− and HO2• were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2•− permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2•− detection in the internalized parasite. Importantly, O2•− detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2•− or during macrophage infections. Other mechanisms of O2•− entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2•− detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB–overexpressing parasites, supporting the role of the cytosolic O2•−-catabolizing enzyme as a virulence factor for CD.

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“…We then confirmed that Grx2 protection against SIN‐1 induced nitration in A2B5 positive cells is limited as well (170 ± 30% and 183 ± 36% with and without Grx2, respectively, Figure S4B, Supporting Information). Utilization of our newly developed peroxynitrite probe fluorescein‐boronate (Rios et al, ) in HeLa cells overexpressing Grx2c revealed intracellular inhibition of ONOO ‐ ‐formation by elimination of • NO as protective Grx2 mechanism decreasing oxidative and nitrosative protein modifications. The amount of ONOO ‐ went down to 27 ± 4% or 56 ± 7% in Grx2c overexpressing cells compared to wildtype cells treated with GSNO or SIN‐1, respectively (Figure e, f).…”

Section: Resultsmentioning

confidence: 99%

“…Measurement of nitric oxide was performed with the Griess Reagent from Molecular Probes according to manufacturer's protocol using a microplate reader (Tecan, Molecular Devices, absorbance at 540 nm). Measurement of peroxynitrite was based on the reaction of fluorescein‐boronate to fluorescein in the presence of peroxynitrite (Rios et al, ). The probe was synthesized and solved in DMSO and further diluted and measured in PBS at a final concentration of 50 µM.…”

Section: Methodsmentioning

confidence: 99%

Iron‐sulfur glutaredoxin 2 protects oligodendrocytes against damage induced by nitric oxide release from activated microglia

Lepka

Volbracht

Bill

et al. 2017

Glia

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Demyelinated brain lesions, a hallmark of autoimmune neuroinflammatory diseases like multiple sclerosis, result from oligodendroglial cell damage. Activated microglia are considered a major source of nitric oxide and subsequent peroxynitrite-mediated damage of myelin. Here, we provide biochemical and biophysical evidence that the oxidoreductase glutaredoxin 2 inhibits peroxynitrite formation by transforming nitric oxide into dinitrosyl-diglutathionyl-iron-complexes. Glutaredoxin 2 levels influence both survival rates of primary oligodendrocyte progenitor cells and preservation of myelin structure in cerebellar organotypic slice cultures challenged with activated microglia or nitric oxide donors. Of note, glutaredoxin 2-mediated protection is not linked to its enzymatic activity as oxidoreductase, but to the disassembly of its uniquely coordinated iron-sulfur cluster using glutathione as non-protein ligand. The protective effect of glutaredoxin 2 is connected to decreased protein carbonylation and nitration. In line, brain lesions of mice suffering from experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, show decreased glutaredoxin 2 expression and increased nitrotyrosine formation indicating that this type of protection is missing in the inflamed central nervous system. Our findings link inorganic biochemistry to neuroinflammation and identify glutaredoxin 2 as a protective factor against neuroinflammation-mediated myelin damage. Thus, improved availability of glutathione-coordinated iron-sulfur clusters emerges as a potential therapeutic approach in inflammatory demyelination.

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Sensitive Detection and Estimation of Cell-Derived Peroxynitrite Fluxes Using Fluorescein-Boronate

Cited by 14 publications

References 0 publications

Tracking isotopically labeled oxidants using boronate-based redox probes

Tracking isotopically labeled oxidants using boronate-based redox probes

Cytosolic Fe-superoxide dismutase safeguardsTrypanosoma cruzifrom macrophage-derived superoxide radical

Iron‐sulfur glutaredoxin 2 protects oligodendrocytes against damage induced by nitric oxide release from activated microglia

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