Introducción

Tenemos el gusto de presentar el tercer suplemento que contiene la selección de las monografías realizadas por los estudiantes del Ciclo de Metodología Científica II, que cursó la generación de sexto año de la carrera de Medicina de la Universidad de la República, en 2016 y el cual, con el objetivo de completar la formación científica, todos los estudiantes realizan un trabajo de investigación a lo largo de todo el año. Dichos trabajos se realizaron en grupos de hasta cinco estudiantes dirigidos por docentes orientadores en todas las áreas disciplinares de la Facultad de Medicina incluyendo básicas, clínicas y epidemiológicas. La publicación de estos trabajos representa la culminación de un esfuerzo académico formativo que fue iniciado por los alumnos en el año 2016 y en el que todos los grupos presentaron su trabajo en forma escrita como monografía científica, y bajo forma de poster en las Jornadas Científicas de Metodología Científica que tuvieron lugar en diciembre del año pasado en la Facultad de Medicina

Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration

In this review we provide an analysis of the biochemistry of peroxynitrite and tyrosine nitration. Peroxynitrite is the product of the diffusion-controlled reaction between superoxide (O2â¢-) and nitric oxide (â¢NO). This process is in competition with the enzymatic dismutation of O2â¢- and the diffusion of â¢NO across cells and tissues and its reaction with molecular targets (e.g. guanylate cyclase). Understanding the kinetics and compartmentalization of the O2â¢- / â¢NO interplay is critical to rationalize the shift of â¢NO from a physiological mediator to a cytotoxic intermediate. Once formed, peroxynitrite (ONOO- and ONOOH; pKa = 6,8) behaves as a strong one and two-electron oxidant towards a series of biomolecules including transition metal centers and thiols. In addition, peroxynitrite anion can secondarily evolve to secondary radicals either via its fast reaction with CO2 or through proton-catalyzed homolysis. Thus, peroxynitrite can participate in direct (bimolecular) and indirect (through secondary radical intermediates) oxidation reactions; through these processes peroxynitrite can participate as cytotoxic effector molecule against invading pathogens and/or as an endogenous pathogenic mediator. Peroxynitrite can cause protein tyrosine nitration in vitro and in vivo. Indeed, tyrosine nitration is a hallmark of the reactions of â¢NO-derived oxidants in cells and tissues and serves as a biomarker of oxidative damage. Protein tyrosine nitration can mediate changes in protein structure and function that affect cell homeostasis. Tyrosine nitration in biological systems is a free radical process that can be promoted either by peroxynitrite-derived radicals or by other related â¢NO-dependent oxidative processes. Recently, mechanisms responsible of tyrosine nitration in hydrophobic biostructures such as membranes and lipoproteins have been assessed and involve the parallel occurrence and connection with lipid peroxidation. Experimental strategies to reveal the proximal oxidizing mechanism during tyrosine nitration in given pathophysiologically-relevant conditions include mapping and identification of the tyrosine nitration sites in specific proteins. Keywords: Free radicals, Oxidants, Nitric oxide, Peroxynitrite and tyrosine nitratio

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/ peroxide-dependent mechanism

41 Pags.- 3 Tabls.- 10 Figs. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-313XProtein Tyr nitration is a post-translational modification yielding 3-nitrotyrosine (NO2-Tyr). Formation of NO2-Tyr is generally considered as a marker of nitroxidative stress and is involved in some human pathophysiological disorders, but it has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2-Tyr in Lbs. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2-Tyr25 and NO2-Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (•NO) and NO2- by alternative means to nitrate reductase, probably via a NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lbs revealed that Tyr nitration requires NO2- + H2O2 and that peroxynitrite is not an efficient inducer of nitration, possibly by isomerizing it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2-Tyr in Lbs is a consequence of active metabolism in functional nodules, where Lbs may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.M.S. was the recipient of a predoctoral contract (Programa Junta de Ampliación de Estudios) from CSIC. L.C.-B. was the recipient of a predoctoral contract (Formación de Personal Investigador) from Ministerio de Economía y Competitividad (MINECO). C.S. was funded by the Austrian Fonds zur Förderung der wis-senschaftlichen Forschung (P23441-B20). This work was mainly supported by MINECO-Fondo Europeo de Desarrollo Regional (AGL2011-24524 and AGL2014-53717-R) and CSIC (Proyecto Intramural Especial 201240E150). Additional funding was provided by grants of Agencia Nacional de Investigación e Innovación (Fondo Clemente Estable, FCE 6605) to S.B. and Universidad de la República and National Institutes of Health (RO1 AI095173) to R.R.Peer reviewe

A computational investigation of the reactions of tyrosyl, tryptophanyl, and cysteinyl radicals with nitric oxide and molecular oxygen

Proteins are main targets of oxidants in biological systems. This oxidation may occur in the protein backbone as well as in certain amino acid side chains, depending on the oxidant and amino acid intrinsic reactivity. Moreover, many enzymes are capable of generating stable amino acid radicals, such as tyrosyl, tryptophanyl and cysteinyl radicals. These species react very rapidly (many times as diffusion-controlled reactions) with relevant cellular open-shell species such as nitric oxide (·NO) or molecular oxygen (O 2 ). The exception to this apparent rule is tyrosyl radical, that reacts at diffusion rates with ·NO, but shows very slow reactivity towards O 2 (rate constant <10 3 M −1 s −1 ). In this work, we provide a comparative molecular-level description of the reaction mechanisms involved in the reactions of tyrosyl, tryptophanyl and cysteinyl radicals towards ·NO and O 2 , through quantum mechanics simulations which allow us to obtain relevant energetic and structural parameters, proposing a molecular explanation to this tyrosyl discrimination capability, namely, its marginal reactivity with O 2 .Fil: Pedron, Federico Nicolás. 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: Bartesaghi, Silvina. 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; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la República; Uruguay. 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

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Funding Information: This work was supported by grants of Universidad de la República (CSIC_2018 and EI_2020) to R. R., Universidad de la República (CSIC Iniciación_2017_ID_159) to N. C., Alexander von Humboldt Foundation (AvHF) to T. G. and R. R., the Novo Nordisk Foundation ( NNF13OC0004294 and NNF20SA0064214 ) to M. J. D., and PICT 2018-0795 from Agencia I+d+i Argentina , University of Buenos Aires (grant 20020120300025BA ), and CONICET (grant 11220150100303CO ) to D. E. N. C. was partially supported by a fellowship from Comisión Académica de Posgrado (CAP), Universidad de la República , Uruguay. Additional funding was obtained from Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay), Agencia Nacional de Investigación e Innovación (ANII-SNI, Uruguay), EU-LAC Health (EULACH16/T01-0131), and Programa de Alimentos y Salud Humana (PAyS, Uruguay). Funding Information: The authors would like to thank Dr Tobias Karlberg and Dr Susanne Gräslund from the Karolinska Institutet - Structural Genomics Consortium for providing the HsGS plasmid (Construct ID GLULA-c004). We also thank Dr Verónica Tórtora for her major aid within the initial steps of the transformation and HsGS expression experiments. N. C. D. E. P. H. T. G. M. J. D. S. B. and R. R. conceptualization; N. C. Mauricio Mastrogiovanni, Michele Mariotti, F. M. I. and P. H. methodology; N. C. Mauricio Mastrogiovanni, Michele Mariotti, and F. M. I. investigation; N. C. writing–original draft; M. J. D. S. B. and R. R. writing–review and editing; R. R. supervision. This work was supported by grants of Universidad de la República (CSIC_2018 and EI_2020) to R. R. Universidad de la República (CSIC Iniciación_2017_ID_159) to N. C. Alexander von Humboldt Foundation (AvHF) to T. G. and R. R. the Novo Nordisk Foundation (NNF13OC0004294 and NNF20SA0064214) to M. J. D. and PICT 2018-0795 from Agencia I+d+i Argentina, University of Buenos Aires (grant 20020120300025BA), and CONICET (grant 11220150100303CO) to D. E. N. C. was partially supported by a fellowship from Comisión Académica de Posgrado (CAP), Universidad de la República, Uruguay. Additional funding was obtained from Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay), Agencia Nacional de Investigación e Innovación (ANII-SNI, Uruguay), EU-LAC Health (EULACH16/T01-0131), and Programa de Alimentos y Salud Humana (PAyS, Uruguay). Publisher Copyright: © 2023 The AuthorsGlutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.publishersversionpublishe

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: Computer simulation studies in model tyrosine-cysteine peptides in solution

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues. © 2012 Elsevier Inc. All rights reserved.Fil: Petruk, Ariel Alcides. 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. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Química del Noroeste. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química del Noroeste; ArgentinaFil: Bartesaghi Hierro, Silvina María. 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Kalyanaraman, Balaraman. Medical College Of Wisconsin; Estados UnidosFil: Marti, Marcelo Adrian. 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Radi, Rafael. Universidad de la República; Urugua

3-Nitrotyrosine and related derivatives in proteins: precursors, radical intermediates and impact in function

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical–radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO−), the product of the reaction of superoxide radical (O2•−) with •NO, but ONOO−-independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.Fil: Campolo, Nicolás. Universidad de la República; UruguayFil: Issoglio, Federico Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: 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: Bartesaghi Hierro, Silvina María. Universidad de la República; UruguayFil: Radi, Rafael. Universidad de la República; Urugua