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

Contenido de oligoelementos y factores antinutricionales de hojas comestibles nativas de Mesoamérica

Los oligoelementos son importantes constituyentes nutricionales de las hierbas comestibles. Se colectaron 11 especies, nueve nativas (Amaranthus hybridus, Cnidoscolus chayamansa, Crotalaria longirostrata, Dysphania ambrosioides, Lycianthes synanthera, Sechium edule, Solanum americanum, S. nigrescens, S. wendlandii) y dos extranjeras (Moringa oleifera, Spinacea oleracea) en dos regiones de Guatemala. Se prepararon muestras de la hierba seca, cocida y de caldo de hierba fresca. Se cuantificaron por espectrofotometría de absorción atómica los macro (N, P, K) y oligoelementos (Ca, Mg, Na, Cu, Zn, Mn), taninos por espectrofotometría y oxalatos por permanganimetría. El contenido de oligoelementos es diverso, para Zn, la hierba control S. oleracea contienen buena cantidad (90-140 ppm); de las nativas D. ambrosioides (130-160 ppm) y A. hybridus (70-80 ppm) tienen la mayor cantidad. Respecto a Fe las hierbas control tienen buena composición (S. oleracea, 220-280 ppm y M. oleifera, 105-135 ppm); de las nativas A. hybridus (90-240 ppm), C. aconitifolius (75-185 ppm) y L. synanthera (75-140 ppm) tienen las mayores concentraciones. Se encontraron niveles elevados de oxalatos en S. oleracea (67.30 (5.51) mg/g), L. synanthera (56.30 (9.67) mg/g) y S. nigrescens (33.6 (5.48) mg/g); en las demás hierbas se encontraron niveles menores. Los niveles de taninos fueron bajos (0.1-0.8 mg/g) para todas las especies. Se demuestra que cuatro especies nativas tienen un buen contenido de oligoelementos y presentan valores menores de antinutricionales que los controle

Immune profiling in Puerto Rican injection drug users with and without HIV-1 infection

Antiretroviral therapy has been effective in suppressing HIV viral load and enabling people living with HIV to experience longer, more conventional lives. However, as people living with HIV are living longer, they are developing aging-related diseases prematurely and are more susceptible to comorbidities that have been linked to chronic inflammation. Coincident with HIV infection and aging, drug abuse has also been independently associated with gut dysbiosis, microbial translocation, and inflammation. Here, we hypothesized that injection drug use would exacerbate HIV-induced immune activation and inflammation, thereby intensifying immune dysfunction. We recruited 50 individuals not using injection drugs (36/50 HIV+) and 47 people who inject drugs (PWID, 12/47 HIV+). All but 3 of the HIV+ subjects were on antiretroviral therapy. Plasma immune profiles were characterized by immunoproteomics, and cellular immunophenotypes were assessed using mass cytometry. The immune profiles of HIV+/PWID−, HIV−/PWID+, and HIV+/PWID+ were each significantly different from controls; however, few differences between these groups were detected, and only 3 inflammatory mediators and 2 immune cell populations demonstrated a combinatorial effect of injection drug use and HIV infection. In conclusion, a comprehensive analysis of inflammatory mediators and cell immunophenotypes revealed remarkably similar patterns of immune dysfunction in HIV-infected individuals and in people who inject drugs with and without HIV-1 infection

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 103 times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pKa reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 108 and 105 M− 1 s− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pKa. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.Fil: Reyes, Aníbal M. Universidad de la República; UruguayFil: Hugo. Martín. Universidad de la República; UruguayFil: Trostchansky, Andrés. Universidad de la República; UruguayFil: Capece, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de Los Materiales, Medioambiente y Energia; ArgentinaFil: Radi, Rafael. Universidad de la República; UruguayFil: Trujillo, Madia. Universidad de la República; Urugua

Mitochondrial Peroxiredoxin 3 Is Rapidly Oxidized and Hyperoxidized by Fatty Acid Hydroperoxides

Human peroxiredoxin 3 (HsPrx3) is a thiol-based peroxidase responsible for the reduction of most hydrogen peroxide and peroxynitrite formed in mitochondria. Mitochondrial disfunction can lead to membrane lipoperoxidation, resulting in the formation of lipid-bound fatty acid hydroperoxides (LFA-OOHs) which can be released to become free fatty acid hydroperoxides (fFA-OOHs). Herein, we report that HsPrx3 is oxidized and hyperoxidized by fFA-OOHs including those derived from arachidonic acid and eicosapentaenoic acid peroxidation at position 15 with remarkably high rate constants of oxidation (>3.5 × 107 M−1s−1) and hyperoxidation (~2 × 107 M−1s−1). The endoperoxide-hydroperoxide PGG2, an intermediate in prostanoid synthesis, oxidized HsPrx3 with a similar rate constant, but was less effective in causing hyperoxidation. Biophysical methodologies suggest that HsPrx3 can bind hydrophobic structures. Indeed, molecular dynamic simulations allowed the identification of a hydrophobic patch near the enzyme active site that can allocate the hydroperoxide group of fFA-OOHs in close proximity to the thiolate in the peroxidatic cysteine. Simulations performed using available and herein reported kinetic data indicate that HsPrx3 should be considered a main target for mitochondrial fFA-OOHs. Finally, kinetic simulation analysis support that mitochondrial fFA-OOHs formation fluxes in the range of nM/s are expected to contribute to HsPrx3 hyperoxidation, a modification that has been detected in vivo under physiological and pathological conditions

MEDICIONES ULTRASONOGRAFICAS Y DE TOMOGRAFIA COMPUTADA DE ADIPOSIDAD Y ALTERACIONES METABOLICAS ASOCIADAS A OBESIDAD EN NIÑOS

Introducción. La obesidad infantil es un problema de salud pública de prevalencia creciente y consecuencias a futuro. El compartimiento adiposo intraperitoneal estaría asociado a factores de riesgo metabólicos propios de la obesidad. No existe una estandarización de mediciones de tejido adiposo en imágenes en niños. Objetivos. Estudiar la asociación entre mediciones de tejido graso abdominal con insulinemia, en niños. Sujetos y Métodos. Se estudiaron 37 escolares prepuberales obesos (IMC ? p95), de ambos sexos, entre 6 y 12 años, con técnicas antropométricas, imagenológicas (US y TC) y de laboratorio (glicemia, insulinemia). Resultados. Las mediciones del tejido adiposo abdominal mediante US presentaron altas correlaciones con las mismas mediciones por TC (r= 0,79; pIntroduction. Childhood obesity is increasing over the world with serious health consequences. Intraabdominal fat is associated to some metabolic alterations in obesity. It does not exist a standard imaging method to measure adipose tissue in children. Objectives: In obese children to study the association between insulinemia and subcutaneous or intraabdominal fat evaluated by ultrasonographic (US) or computed tomography (CT). Subjects and Methods. 37 obese (BMI ? p95) prepubertal obese children (ages from 6 to 12 years) were assessed using anthropometric, US and CT for fat areas and linear intrabdominal segments measurements. Laboratory techniques were also performed: insulinemia and glycemia. Results. We found good correlations between US and TC intra-abdominal adipose tissue measurements (r= 0,79; p< 0,001). US (r=0.56) and CT (r=0.53) visceral fat assessment had better correlations with insulinemia than anthropometric measurements (BMI, r= 0,33; waist, r= 0.42). Conclusions. US and CT measurements of intraabdominal fat emerge as promising techniques to identify obesity-associated metabolic risk in childhoo

Ultrasonographic and by computed tomography measurements of adipose tissue and metabolic changes associated with obesity in children Mediciones ultrasonograficas y de tomografia computada de adiposidad y alteraciones metabolicas asociadas a obesidad en ni

Introduction. Childhood obesity is increasing over the world with serious health consequences. Intraabdominal fat is associated to some metabolic alterations in obesity. It does not exist a standard imaging method to measure adipose tissue in children. Objectives: In obese children to study the association between insulinemia and subcutaneous or intraabdominal fat evaluated by ultrasonographic (US) or computed tomography (CT). Subjects and Methods. 37 obese (BMI ? p95) prepubertal obese children (ages from 6 to 12 years) were assessed using anthropometric, US and CT for fat areas and linear intrabdominal segments measurements. Laboratory techniques were also performed: insulinemia and glycemia. Results. We found good correlations between US and TC intra-abdominal adipose tissue measurements (r= 0,79; p&lt; 0,001). US (r=0.56) and CT (r=0.53) visceral fat assessment had better correlations with insulinemia than anthropometric measurements (BMI, r= 0,33; waist, r= 0.42). Conclusions. US an

The role of nitric oxide signaling in pulmonary circulation of high- and low-altitude newborn sheep under basal and acute hypoxic conditions

Nitric oxide (NO) is the main vasodilator agent that drives the rapid decrease of pulmonary vascular resistance for the respiratory onset during the fetal to neonatal transition. Nevertheless, the enhanced NO generation by the neonatal pulmonary arterial endothelium does not prevent development of hypoxic pulmonary hypertension in species without an evolutionary story at high altitude. Therefore, this study aims to describe the limits of the NO function at high-altitude during neonatal life in the sheep as an animal model without tolerance to perinatal hypoxia. We studied the effect of blockade of NO synthesis with L-NAME in the cardiopulmonary response of lowland (580 m) and highland (3600 m) newborn lambs basally and under an episode of acute hypoxia. We also determined the pulmonary expression of proteins that mediate the actions of the NO vasodilator pathway in the pulmonary vasoactive tone and remodeling. We observed an enhanced nitrergic function in highland lambs under basal conditions, evidenced as a markedly greater increase in basal mean pulmonary arterial pressure (mPAP) and resistance (PVR) under blockade of NO synthesis. Further, acute hypoxic challenge in lowland lambs infused with L-NAME markedly increased their mPAP and PVR to values greater than baseline, whilst in highland animals under NO synthesis blockade, these variables did not show additional increase in response to low PO2. Highland animals showed increased pulmonary RhoA expression, decreased PSer188-RhoA fraction, increased PSer311-p65-NFқβ fraction and up-regulated smooth muscle α-actin, relative to lowland controls. Taken together our data suggest that NO-mediated vasodilation is important to keep a low pulmonary vascular resistance under basal conditions and acute hypoxia at low-altitude. At high-altitude, the enhanced nitrergic signaling partially prevents excessive pulmonary hypertension but does not protect against acute hypoxia. The decreased vasodilator efficacy of nitrergic tone in high altitude lambs could be in part due to increased RhoA signaling that opposes to NO action in the hypoxic pulmonary circulation

Mycothiol/mycoredoxin 1-dependent reduction of the peroxiredoxin AhpE from mycobacterium tuberculosis

Background: Mycothiol, the major low-molecular weight thiol of Mycobacterium tuberculosis, is important for peroxide detoxification and virulence. Results: Mycothiol, together with mycoredoxin-1, a glutaredoxin-like protein, reduces the one-cysteine peroxiredoxin AhpE from the bacterium. Conclusion: Mycobacterium tuberculosis AhpE is a mycothiol/mycoredoxin-1- dependent peroxidase. Significance: Our results provide the first molecular link between a thiol-dependent peroxidase and the mycothiol/mycoredoxin- 1 pathway in Mycobacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc