Potential modulation of sirtuins by oxidative stress

Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1-7), all of them sharing a conserved catalytic core and showing differential subcellular localization and activities. Oxidative stress can affect the activity of sirtuins at different levels: expression, posttranslational modifications, protein-protein interactions, and NAD levels. Mild oxidative stress induces the expression of sirtuins as a compensatory mechanism, while harsh or prolonged oxidant conditions result in dysfunctional modified sirtuins more prone to degradation by the proteasome. Oxidative posttranslational modifications have been identified in vitro and in vivo, in particular cysteine oxidation and tyrosine nitration. In addition, oxidative stress can alter the interaction with other proteins, like SIRT1 with its protein inhibitor DBC1 resulting in a net increase of deacetylase activity. In the same way, manipulation of cellular NAD levels by pharmacological inhibition of other NAD-consuming enzymes results in activation of SIRT1 and protection against obesity-related pathologies. Nevertheless, further research is needed to establish the molecular mechanisms of redox regulation of sirtuins to further design adequate pharmacological interventions

Permeability of human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a 64th Annual Meeting of the Biophysical Society, San Diego, CA. 2020Hydrogen peroxide (H2O2) and other reactive species are important physiological mediators in the vascular system. Enzymatic production of H2O2 is involved in regulating cell growth, proliferation and vasodilation. Whereas endothelial cells are important sources of H2O2, red blood cells (RBC) are considered the most important sinks of H2O2 in the vasculature. However, little is known about the permeability of their membrane to H2O2. The permeability coefficient of human RBC membranes to H2O2 was determined using the enzyme latency method, based on measuring the rate of H2O2 decomposition in lysed vs whole cells. If the passage through the membrane is the rate limiting step in H2O2 decomposition, then a difference is observed that can be used to calculate the permeability coefficient. Additional experiments were done to differentiate between simple diffusion through the lipid fraction and facilitated diffusion through protein channels. The lack of reported permeability coefficients for lipid membranes prompted us to do experiments with phospholipid-cholesterol liposome membranes that indicated that simple diffusion is a slow process. Determination of partition coefficients in different solvents mimicking different depths of the membrane indicate that the low permeability of lipid membranes to H2O2 is caused mainly by its very low solubility in the acyl region of the bilayer. The activation energy of permeation through RBC membranes suggested that protein channels were involved in facilitating H2O2 diffusion through the membrane. Inhibitors of hAQP3 and hAQP1 had no effect in H2O2 consumption rate, suggesting that other membrane proteins may be involved. Although the RBC membrane presents a significant barrier to H2O2 passage, especially in comparison with other solutes such as oxygen and nitric oxide, the permeability is still high enough to support the role of RBC as sinks of H2O2 in circulation.ANII: FMV_1_2019_15559

Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2

Peroxiredoxins are thiol-dependent peroxidases that function in peroxide detoxification and H2O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H2O2 and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H2O2 than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H2O2 with rate constants of ca 2 × 103 M−1 s−1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s−1 for PRDX1, 0.2 s−1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.Fil: Dalla Rizza, Joaquín. Universidad de la Republica; UruguayFil: Randall, Lía M.. Universidad de la Republica; UruguayFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferrer Sueta, Gerardo. Universidad de la Republica; UruguayFil: Denicola, Ana. Universidad de la República; Urugua

Detection and quantification of nitric oxide–derived oxidants in biological systems

The free radical nitric oxide (NO) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO -derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO , and we discuss the approaches currently available for the analysis of NO , nitrogen dioxide (NO2 ), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO /ONOOH), as well as peroxynitrite-derived hydroxyl (HO) and carbonate anion (CO3) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2), nitrate (NO3), nitrosyl–metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state– of–the–art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO -derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involveAgencia Nacional de Investigación e Innovación FCE_1_2017_1_136043Comisión Sectorial de Investigación Científica (CSIC)Universidad de la República. Espacio Interdisciplinari

Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols

Life on Earth evolved in the presence of hydrogen peroxide, and other peroxides also emerged before and with the rise of aerobic metabolism. They were considered only as toxic byproducts for many years. Nowadays, peroxides are also regarded as metabolic products that play essential physiological cellular roles. Organisms have developed efficient mechanisms to metabolize peroxides, mostly based on two kinds of redox chemistry, catalases/peroxidases that depend on the heme prosthetic group to afford peroxide reduction and thiol-based peroxidases that support their redox activities on specialized fast reacting cysteine/selenocysteine (Cys/Sec) residues. Among the last group, glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) are the most widespread and abundant families, and they are the leitmotif of this review. After presenting the properties and roles of different peroxides in biology, we discuss the chemical mechanisms of peroxide reduction by low molecular weight thiols, Prxs, GPxs, and other thiol-based peroxidases. Special attention is paid to the catalytic properties of Prxs and also to the importance and comparative outlook of the properties of Sec and its role in GPxs. To finish, we describe and discuss the current views on the activities of thiol-based peroxidases in peroxide-mediated redox signaling processes.Fil: Zeida, Ari. Universidad de la República; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Ferrer Sueta, Gerardo. Universidad de la Republica; UruguayFil: Denicola, Ana. Universidad de la Republica; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Radi, Rafael. Universidad de la República; Urugua

Leukoreduction of red blood cell concentrates for transfusion

Resumen del Conference Paper presentado a SfRBM 27th Annual Conferenc

Novel antiprotozoal products: Imidazole and benzimidazole N-oxide derivatives and related compounds

The syntheses and biological evaluation of the first anti-protozoa imidazole N-oxide and benzimidazole N-oxide and their derivatives are reported. They were tested in vitro against two different protozoa, Trypanosoma cruzi and Trichomonas vaginalis. Derivative 7c, ethyl-1-(i-butyloxycarbonyloxy)-6-nitrobenzimid-azole-2-carboxylate, displayed activity on both protozoa. Lipophilicity and redox potential were experimentally determined in order to study the relationship with activity of the compounds. These properties are well related with the observed bioactivity. Imidazole and benzimidazole N-oxide derivatives are becoming leaders for further chemical modifications and advanced biological studies

Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.MEC: I/FVF2017/069ANII: FCE_1_2017_1_136043CSIC: I+D 2017; I+D 202