Evolution of the ¨Oxidative Stress¨ concept: half of a century contributions from the Facultad de Medicina, Montevideo, Uruguay

Rafael Radi: Departamento de Bioquímica y Centro de Investigaciones Biomédicas. Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800, Montevideo, Uruguay. Nombrado Doctor Honoris Causa de la Facultad de Medicina, Universidad de la República, Uruguay en 2010.Contacto: Rafael Radi. E-mail: [email protected] siguiente revisión presenta algunos elementos acerca de la evolución del concepto de “Estrés Oxidativo”, que van desde las primeras observaciones bioquímicas en sistemas enzimáticos hasta aproximaciones terapéuticas actuales a nivel preclínico. En el transcurso de ya más de medio siglo de aportes, se destaca en etapas tempranas del desarrollo del concepto la participación de investigadores que trabajaron activamente en la Facultad de Medicina, Universidad de la República (Montevideo, Uruguay) y en particular en su Departamento de Bioquímica, incluyendo al Profesor Visitante John Totter y al Profesor Eugenio Prodanov. El entendimiento de los procesos metabólicos que conducen a la formación y eliminación de especies oxidantes y radicales libres, y el desarrollo de técnicas para su identificación, detección y cuantificación ha sido un foco central del área. Se analiza el desarrollo y evolución del concepto de “Estrés Oxidativo” que se define actualmente como una situación de desbalance metabólico que conduce a alteración de la señalización redox y/o daño oxidativo facilitando el desarrollo y progresión de patología degenerativa e inflamatoria. Los fenómenos oxidativos también son utilizados por células del sistema inmune como mecanismo citotóxico contra patógenos invasores. Un avance importante del área se dio con el reconocimiento que el metabolismo del radical óxido nítrico se conecta con procesos oxidativos, en particular a través de su reacción difusional con el radical superóxido para rendir peroxinitrito, un peróxido y nucleófilo inestable y reactivo en sistemas biológicos. La mejor comprensión de los mecanismos intra y extracelulares de formación de especies oxidantes, incluyendo el rol de la disfunción mitocondrial, y los mecanismos de señalización redox, están dando lugar al desarrollo de estrategias nutricionales y farmacológicas con capacidad de modular in vivo fenómenos oxidativos asociados a patología. He seleccionado observaciones y trabajos con énfasis bioquímico que considero relevantes a nivel universal, relacionándolos e integrándolos con aportes que se han realizado desde nuestra Facultad en el área de estrés oxidativo.The following review presents elements in relation to the evolution of the “Oxidative Stress” concept, that span from the first biochemical observations in enzymatic systems to the current therapeutic strategies at the preclinical level. In more than half a century of contributions to the field, it is remarkable the participation of investigators of the Facultad de Medicina, Universidad de la República (Montevideo, Uruguay) from the early days of the research, in particular the Department of Biochemistry, including Visiting Professor John Totter and Professor Eugenio Prodanov. The understanding of the metabolic processes that lead to the formation and detoxification of free radicals and oxidant species, and the development of methodologies for its identification, detection and quantitation has been a central topic of the area. The concept of oxidative stress has developed and evolved to the current definition representing a metabolic imbalance that leads to the alteration of redox signaling and/or an oxidative damage, which in turn facilitates the development and progression of degenerative and inflammatory pathology. Oxidative events are also utilized by immune system cell as a cytotoxic mechanism against invading pathogens. An important advance in the area was the recognition that the metabolism of the free radical nitric oxide was connected with oxidative processes, in particular through its diffusional reaction with superoxide radical to yield peroxynitrite, an unstable and reactive nucleophile and peroxide in biological systems. A better understanding of the intra- and extracellular mechanisms of formation of oxidant species, including the role of mitochondrial dysfunction, as well as the mechanisms of redox signaling, are providing opportunities for developing nutritional and pharmacological strategies for the in vivo modulation of oxidative events associated to pathology. I have selected observations and works with a biochemical emphasis that I consider relevant, and relating and integrating them with contributions carried out from our Faculty in the area of oxidative stress

The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics

Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The pK a alk values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of pK a alk is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the pK a alk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pK a alk upshift. The different physicochemical conditions found here to influence pK a alk are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo.Fil: Oviedo Rouco, Santiago. 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: Castro, Maria Ana. 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: Álvarez Paggi, Damián Jorge. 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: Spedalieri, Ana Cecilia. 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: Tortora, Verónica. Universidad de la República; UruguayFil: Tomasina, Florencia. Universidad de la República; UruguayFil: Radi, Rafael. Universidad de la República; UruguayFil: Murgida, Daniel Horacio. 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; Argentin

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

Specific methionine oxidation of cytochrome c in complexes with zwitterionic lipids by hydrogen peroxide: potential implications for apoptosis

Cytochrome c (Cyt-c) has been previously shown to participate in cardiolipin (CL) oxidation and, therefore,in mitochondrial membrane permeabilization during the early events of apoptosis. The gain in this function has been ascribed to specific CL/Cyt-c interactions. Here we report that the cationic protein Cyt-c is also able to interact electrostatically with the main lipid components of the mitochondrial membranes, the zwitterionic lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), through the mediation of phosphate anions that bind specifically to amino groups in the surfaces of protein and model membranes. In these complexes, Cyt-c reacts efficiently with H2O2 at submillimolar levels, which oxidizes the sulfur atom of the axial ligand Met80. The modified protein is stable and presents significantly enhanced peroxidatic activity. Based on these results, we postulate that the rise of H2O2 concentrations to the submillimolar levels registered during initiation of the apoptotic program may represent one signaling event that triggers the gain in peroxidatic function of the Cyt-c molecules bound to the abundant PE and PC membrane components. As the activated protein is a chemically stable species, it can potentially bind and oxidize important targets, such as CL.Fil: Capdevila, Daiana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Departamento de Ciencias Exactas; ArgentinaFil: Marmisollé, Waldemar Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Departamento de Ciencias Exactas; ArgentinaFil: Tomasina, Florencia. Universidad de la Republica. Facultad de Medicina; UruguayFil: Demicheli, Verónica. Universidad de la Republica. Facultad de Química y Facultad de Ciencias; UruguayFil: Portela, Magdalena . Instituto Pasteur de Montevideo; UruguayFil: Radi, Rafael . Universidad de la Republica. Facultad de Química y Facultad de Ciencias; UruguayFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; Argentin

The extraordinary catalytic ability of peroxiredoxins: a combined experimental and QM/MM study on the fast thiol oxidation step

Peroxiredoxins (Prxs) catalyze the reduction of peroxides, a process of key relevance in a variety of cellular processes. The first step in the catalytic cycle of all Prxs is the oxidation of a cysteine residue to sulfenic acid, which occurs 103–107 times faster than in free cysteine. We present an experimental kinetics and hybrid QM/MM investigation to explore the reaction of Prxs with H2O2 using alkyl hydroperoxide reductase E from Mycobacterium tuberculosis as a Prx model. We report for the first time the thermodynamic activation parameters of H2O2 reduction using Prx, which show that protein significantly lowers the activation enthalpy, with an unfavourable entropic effect, compared to the uncatalyzed reaction. The QM/MM simulations show that the remarkable catalytic effects responsible for the fast H2O2 reduction in Prxs are mainly due to an active-site arrangement, which establishes a complex hydrogen bond network activating both reactive species.Fil: Zeida Camacho, Ari Fernando. 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Reyes, Aníbal M.. Universidad de la República; UruguayFil: González Lebrero, Mariano Camilo. 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Radi Isola, Rafael. Universidad de la República; UruguayFil: Trujillo, Madia. Universidad de la República; 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; Argentin

Design, Synthesis and Biological Characterization of Potential Antiatherogenic Nitric Oxide Releasing-Tocopherol Analogs.

PostprintSynthesis and biological characterization of a series of tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties and antiplatelet activity. They were also capable to prevent LDL oxidation

Structural basis of redox-dependent modulation of galectin-1 dynamics and function

Galectin-1 (Gal-1), a member of a family of multifunctional lectins, plays key roles in diverse biological processes including cell signaling, immunomodulation, neuroprotection and angiogenesis. The presence of an unusual number of six cysteine residues within Gal-1 sequence prompted a detailed analysis of the impact of the redox environment on the functional activity of this lectin. We examined the role of each cysteine residue in the structure and function of Gal-1 using both experimental and computational approaches. Our results show that: (i) only three cysteine residues present in each carbohydrate recognition domain (CRD) (Cys2, Cys16 and Cys88) were important in protein oxidation, (ii) oxidation promoted the formation of the Cys16-Cys88 disulfide bond, as well as multimers through Cys2, (iii) the oxidized protein did not bind to lactose, probably due to poor interactions with Arg48 and Glu71, (iv) in vitro oxidation by air was completely reversible and (v) oxidation by hydrogen peroxide was relatively slow (1.7 ± 0.2 M(-1) s(-1) at pH 7.4 and 25°C). Finally, an analysis of key cysteines in other human galectins is also provided in order to predict their behaviour in response to redox variations. Collectively, our data provide new insights into the structural basis of Gal-1 redox regulation with critical implications in physiology and pathology.Fil: Guardia, Carlos Manuel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; ArgentinaFil: Caramelo, Julio Javier. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Trujillo, Madia . Universidad de la Republica; UruguayFil: Mendez Huergo, Santiago Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Radi, Rafael . 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; ArgentinaFil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Increased mitochondrial activity upon CatSper channel activation is required for mouse sperm capacitation

To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception