Mechanistics insights of hydrogen peroxide transport through PIP aquaporins pore

Hydrogen peroxide (H2O2) is transported through membranes by aquaporins (AQP). In particular, some plant PIP aquaporins isoforms are efficient H2O2 channels. As water and H2O2 share physicochemical features, it was first supposed that all AQP that transport water could act as an H2O2 channel. However, experimental evidence showed that not all PIP that transport water can transport H2O2. So, the mechanism of H2O2 transport is still an unsolved issue for AQP channels. MtPIP2,3 is a plasma membrane AQP from the legume Medicago truncatula that permeates H2O2. To understand the structural and chemical selectivity mechanisms leading to H2O2 permeability in PIPs, we characterized the particularities of H2O2 passingthrough MtPIP2,3 pore by 1 μs atomistic molecular dynamic simulations. As PIPs are tetrameric pH gated channels we constructed homology MtPIP2,3 models in open and closed states, and with or without H2O2. All models were conformationally stable along the simulation and H2O2 permeation events were found in the simulations in the presence of this molecule. We find that: i- H2O2 molecules can cross the pore in a single file, iidihedral angles adopted by H2O2 along the pore Z axis present a different distribution compared to the angles visited in the solution; in the selectivity-determining NPA region, H2O2 adopts the wider range of dihedral angles, iii- higher residence times are located around the selectivity filter zone in the open channel and moves to the cytoplasmic filterarea in the closed channel; and iv- the constriction in the cytoplasmic filter area seems to be more stringent for H2O2 passage than for water.Our results shed light onto the molecular mechanism of H2O2 passage through MtPIP2,3 and represent the first steps to understand the structural determinants of AQP differential selectivity for these molecules and water.Fil: Chevriau, Jonathan. 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: Zerbetto de Palma, Gerardo Gabriel. 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: Zeida, Ari. Universidad de la República; UruguayFil: Alleva, Karina Edith. 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; ArgentinaXLIX Reunión Anual de la Sociedad Argentina de BiofísicaArgentinaSociedad Argentina de Biofisic

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

Possible molecular basis of the biochemical effects of cysteine-derived persulfides

Persulfides (RSSH/RSS−) are species closely related to thiols (RSH/RS−) and hydrogen sulfide (H2S/HS−), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed

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

Acidity and nucleophilic reactivity of glutathione persulfide

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.Fil: Benchoam, Dayana. Universidad de la República; UruguayFil: Semelak, Jonathan Alexis. 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: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Grassano, Juan S.. Universidad de Buenos Aires; ArgentinaFil: Ferrer-Sueta, Gerardo. Universidad de la Republica; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la Republica; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Möller, Matías N.. 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: Alvarez, Beatriz. Universidad de la Republica; Urugua

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.Fil: Freeman, Samuel L.. University of Bristol; Reino UnidoFil: Skafar, Vera. Universidad de la República; UruguayFil: Kwon, Hanna. University of Leicester; Reino UnidoFil: Fielding, Alistair J.. Liverpool John Moores University; Reino UnidoFil: Moody, Peter C.E.. University of Leicester; Reino UnidoFil: Martínez, Alejandra. 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; Argentina. Universidade Nova de Lisboa; PortugalFil: Inchausti, Lucas. Universidad de la Republica; Uruguay. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Smircich, Pablo. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay. Universidad de la Republica; UruguayFil: Zeida, Ari. Universidad de la Republica; UruguayFil: Piacenza, Lucía. Universidad de la Republica; UruguayFil: Radi, Rafael. Universidad de la Republica; UruguayFil: Raven, Emma L.. University of Bristol; Reino Unid

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and (3)H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379–Cys432 and Cys391–Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold

Structure and reaction mechanism of peroxiredoxins: a theoretical and experimental study

Las peroxirredoxinas (Prxs) constituyen una familia de proteínas encargadas de catalizar la reducción de peróxidos, con función antioxidante y de señalización redox, entre otras. Estas enzimas se encuentran ampliamente distribuidas entre los organismos procariotas y eucariotas, y están usualmente presentes en altas concentraciones. Todas ellas comparten el mismo primer paso en su mecanismo catalítico básico, en el cual la cisteína del sitio activo se oxida a ácido sulfénico por sustratos peróxidos tales como peróxido de hidrógeno (H2O2), peroxinitrito, y una gran variedad de hidroperóxidos orgánicos, lo que ocurre con constantes de velocidad varios órdenes de magnitud mayores que para cisteína libre. Particularmente, la alquil hidroperóxido reductasa E (AhpE) de Mycobacterium tuberculosis es una Prx que reacciona más rápido con peroxinitrito que con H2O2 y que presenta una reactividad particularmente alta frente a hidroperóxidos derivados de ácidos grasos. El presente trabajo de tesis aporta información microscópica acerca de los mecanismos de reducción de peróxidos mediada por tioles, tanto en solución acuosa como en el contexto de la reacción catalizada por Prxs. Para tal fin se emplearon una combinación de técnicas de simulaciones computacionales clásicas, cuánticas e híbridas cuántico-clásicas, en combinación con estudios experimentales de cinética rápida de flujo detenido y de equilibrio. En primer lugar se estudiaron las reacciones de oxidación de tioles de bajo peso molecular por H2O2 y peroxinitrito en solución acuosa, determinando los parámetros termodinámicos de activación y describiendo a nivel molecular el proceso reactivo. Posteriormente se investigaron los determinantes moleculares de la capacidad catalítica de Prxs, utilizando como modelo de trabajo la MtAhpE. En último lugar se analizó la especificidad por el sustrato oxidante para el caso particular de la MtAhpE, comparando la interacción y reactividad con H2O2 y diferentes hidroperóxidos orgánicos.Peroxiredoxins (Prxs) constitute a protein family that catalyzes the reduction of peroxides, with antioxidant and redox signaling functions, among others. These enzymes are ubiquitous and widely distributed in prokaryote and eukaryote organisms, and are usually found at high concentrations. They all share the same first step of their basic catalytic mechanism; the cysteine residue in the active-site gets oxidized by peroxides such as hydrogen peroxide (H2O2), peroxynitrite and a variety of organic hydroperoxides, which occurs several orders of magnitude faster than in the case of free cysteine. In particular, alkyl hydroperoxide reductase (AhpE) from Mycobacterium tuberculosis is a Prx that reacts faster with peroxynitrite than with H2O2 and shows a particularly high reactivity versus fatty acid derived hydroperoxides. The present thesis work is focused in providing molecular insights about peroxide reduction mechanisms by thiols, both in aqueous solution and in the context of the catalyzed reaction by Prxs. For this purpose, a combination of classical, quantum and hybrid quantum-classical simulations, together with fast stopped-flow kinetics and equilibrium experiments was performed. First, we studied the reactions of low molecular weight thiols with H2O2 and peroxynitrite in aqueous solution, determining the thermodynamic activation parameters and describing the reactive process at the molecular level. Afterwards, we investigated the molecular basis of Prxs catalytic ability, using MtAhpE as a model. Finally, the substrate specificity of MtAhpE was analyzed via the comparison of the interaction and reactivity with H2O2 and different organic hydroperoxides.Fil:Zeida Camacho, Ari F.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina