Browsing by Author "Figueroa Alegría, Juan David"
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- ItemAzocompounds as generators of defined radical species: Contributions and challenges for free radical research(2020) López Alarcón, Camilo Ignacio; Fuentes Lemus, Eduardo Felipe; Figueroa Alegría, Juan David; Dorta, Eva; Schöneich, Christian; Davies, Michael J.Peroxyl radicals participate in multiple processes involved in critical changes to cells, tissues, pharmacueticals and foods. Some of these reactions explain their association with degenerative pathologies, including cardiovascular and neurological diseases, as well as cancer development. Azocompounds, and particularly AAPH (2,2 ' Azobis(2-methylpropionamidine) dihydrochloride), a cationic water-soluble derivative, have been employed extensively as sources of model peroxyl radicals. A considerable number of studies have reported mechanistic data on the oxidation of biologically-relevant targets, the scavenging activity of foods and natural products, and the reactions with, and responses of, cultured cells. However, despite the (supposed) experimental simplicity of using azocompounds, the chemistry of peroxyl radical production and subsequent reactions is complicated, and not always considered in sufficient depth when analyzing experimental data. The present work discusses the chemical aspects of azocompounds as generators of peroxyl (and other) radicals, together with their contribution to our understanding of biochemistry, pharmaceutical and food chemistry research. The evidence supporting a role for the formation of alkoxyl (RO center dot) and other radicals during thermal and photochemical decomposition of azocompounds is assessed, together with the potential influence of such species on the reactions under study.
- ItemFormation and characterization of crosslinks, including Tyr-Trp species, on one electron oxidation of free Tyr and Trp residues by carbonate radical anion(2020) Figueroa Alegría, Juan David; Zarate Méndez, Ana María; Fuentes Lemus, Eduardo Felipe; Davies, M. J.; Lopez Alarcon Camilo IgnacioDityrosine and ditryptophan bonds have been implied in protein crosslinking. This is associated with oxidative stress conditions including those involved in neurodegenerative pathologies and age-related processes. Formation of dityrosine and ditryptophan derives from radical-radical reactions involving Tyr(center dot) and Trp(center dot) radicals. However, cross reactions of Tyr(center dot) and Trp(center dot) leading to Tyr-Trp crosslinks and their biological consequences have been less explored. In the present work we hypothesized that exposure of free Tyr and Trp to a high concentration of carbonate anion radicals (CO3 center dot-), under anaerobic conditions, would result in the formation of Tyr-Trp species, as well as dityrosine and ditryptophan crosslinks. Here we report a simple experimental procedure, employing CO3 center dot- generated photochemically by illumination of a Co(iii) complex at 254 nm, that produces micromolar concentrations of Tyr-Trp crosslinks. Analysis by mass spectrometry of solutions containing only the individual amino acids, and the Co(iii) complex, provided evidence for the formation ofo,o '-dityrosine and isodityrosine from Tyr, and three ditryptophan dimers from Trp. When mixtures of Tyr and Trp were illuminated in an identical manner, Tyr-Trp crosslinks were detected together with dityrosine and ditryptophan dimers. These results indicate that there is a balance between the formation of these three classes of crosslinks, which is dependent on the Tyr and Trp concentrations. The methods reported here allow the generation of significant yields of isolated Tyr-Trp adducts and their characterization. This technology should facilitate the detection, and examination of the biological consequences of Tyr-Trp crosslink formation in complex systems in future investigations.
- ItemImplications of differential peroxyl radical-induced inactivation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase for the pentose phosphate pathway(Nature Research, 2022) Reyes Valenzuela, Juan Sebastián; Figueroa Alegría, Juan David; Martínez Rojas, Francisco Javier; López Alarcon, Camilo Ignacio; Fuentes Lemus, Eduardo Felipe; Hagglund, P.M.; Davies, Michael J.; Fierro Huerta, Angelica María; Arenas, Felipe© 2022, The Author(s).Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO·) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2?-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO· source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO·, with Trp and Met being major targets. LC–MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO·-mediated inactivation.
- ItemProtein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times(2020) Cortés Ríos, Javiera Alejandra; Zárate Méndez, Ana María; Figueroa Alegría, Juan David; Medina, J.; Fuentes Lemus, Eduardo Felipe; Rodríguez Fernández, María; Aliaga Miranda, Margarita Elly; López Alarcón, Camilo Ignacio
- ItemQuantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching(2019) Figueroa Alegría, Juan David; Fuentes Lemus, Eduardo Felipe; Dorta Pérez, Eva; Melin, Victoria; Cortés Ríos, Javiera Alejandra; Faúndez Cáceres, Mario; Contreras, David; Denicola, Ana; Álvarez, Beatriz; Davies, Michael J.; López Alarcón, Camilo IgnacioCarbonate radicals (CO3radical dot-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3radical dot- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3radical dot- was followed by visible spectrophotometry. Solutions containing PGR (5–200 μM), SOD-1 (0.3–3 μM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3radical dot- release of 24.6 ± 4.3 μM min−1 for 3 μM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ± 0.1 μM min−1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3radical dot- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3radical dot- release from bSOD-1 and hSOD-1 peroxidase activity.
- ItemRol de triptófano y tirosina en las modificaciones oxidativas de glucosa-6-fosfato deshidrogenasa inducidas por radicales peroxilo y anión radical carbonato(2022) Figueroa Alegría, Juan David; López Alarcón, Camilo Ignacio; Pontificia Universidad Católica de Chile. Escuela de QuímicaLa enzima Glucosa-6-fosfato deshidrogenasa (G6PD) cumple un rol importante en sistemas biológicos. Es la primera enzima de la ruta de las pentosas fosfato, vía fundamental para la síntesis de nucleótidos y para la producción de nicotinamida adenina dinucleótido fosfato reducido (NADPH), cofactor clave para mantener la homeostasis redox intracelular. Aunque se ha sugerido en sistemas biológicos una relación entre los niveles de especies reactivas (ERO) y la actividad de G6PD, son pocos los estudios que han investigado la oxidación de esta enzima y sus consecuencias funcionales. En el presente trabajo de tesis se planteó como hipótesis que las modificaciones oxidativas sobre G6PD, mediadas por radicales peroxilo (ROOl) y radical anión carbonato (CO3l–), dependen de la dosis de radicales y de la disponibilidad de residuos susceptibles a la oxidación. Tales procesos afectarían la actividad catalítica e inducirían cambios en la masa molecular de la proteína, con participación de Trp y Tyr en procesos de entrecruzamiento. En función de dicha hipótesis, se estudió la oxidación de aminoácidos libres (Trp y Tyr) y G6PD, con especial énfasis en la formación de especies di-Tyr, di-Trp y Tyr-Trp, como también en los mecanismos de inactivación de la enzima. Se desarrollaron metodologías por espectrometría de masas (UPLC-MS/MS) para detectar las especies entrecruzadas y se establecieron condiciones experimentales para modular la dosis de los radicales libres. Los resultados obtenidos demostraron que la exposición de Trp y Tyr a diferentes flujos radicalarios no mostró una relación con la formación de di-Tyr y di-Trp. En condiciones experimentales similares, la oxidación de G6PD se asoció con pérdida de la actividad enzimática mediante un proceso dependiente de la dosis, pero no del flujo radicalario. Se determinó una pérdida de 40% de actividad después de la incubación de la enzima con 60 mM de AAPH por 90 minutos. La inactivación de G6PD se relacionó con cambios en su contenido de estructura secundaria como también con un aumento de bolsillos hidrofóbicos. Estos fenómenos indicarían desplegamiento proteico probablemente relacionado con la oxidación de residuos específicos de Met y Trp. Esto último demostrado a través de experimentos desarrollados empleando cloramina T, un oxidante no biológico, específico de residuos de Met. Los resultados obtenidos permiten plantear que la exposición a oxidantes de G6PD conduce a la pérdida de su actividad catalítica lo que puede ser relevante a nivel celular. No obstante, se requieren nuevas investigaciones que permitan establecer, bajo condiciones celulares, la importancia de tales procesos.
- ItemRole of amino acid oxidation and protein unfolding in peroxyl radical and peroxynitrite-induced inactivation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides(2022) Figueroa Alegría, Juan David; Fuentes Lemus, Eduardo Felipe; Reyes Valenzuela, Juan Sebastián; Loaiza Hernández, Matías Ignacio; Aliaga Miranda, Margarita Elly; Fierro Huerta, Angélica; Leinisch, Fabian; Hagglund, Per; Davies, Michael J.; López Alarcón, Camilo IgnacioThe mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO center dot) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride), used as ROO center dot source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaph-thalene-8-sulfonic acid). Incubation of G6PDH (54.4 mu M) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to similar to 59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with similar to 46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO center dot, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO center dot. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.
- ItemThe enzymes of the oxidative phase of the pentose phosphate pathway as targets of reactive species: consequences for NADPH production(2023) Fuentes Lemus, Eduardo Felipe; Reyes Valenzuela, Juan Sebastián; Figueroa Alegría, Juan David; Davies, Michael J.; López Alarcón, Camilo IgnacioThe pentose phosphate pathway (PPP) is a key metabolic pathway. The oxidative phase of this process involves three reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydrogenase (6PGDH) enzymes. The first and third steps (catalyzed by G6PDH and 6PGDH, respectively) are responsible for generating reduced nicotinamide adenine dinucleotide phosphate (NAPDH), a key cofactor for maintaining the reducing power of cells and detoxification of both endogenous and exogenous oxidants and electrophiles. Despite the importance of these enzymes, little attention has been paid to the fact that these proteins are targets of oxidants. In response to oxidative stimuli metabolic pathways are modulated, with the PPP often up-regulated in order to enhance or maintain the reductive capacity of cells. Under such circumstances, oxidation and inactivation of the PPP enzymes could be detrimental. Damage to the PPP enzymes may result in a downward spiral, as depending on the extent and sites of modification, these alterations may result in a loss of enzymatic activity and therefore increased oxidative damage due to NADPH depletion. In recent years, it has become evident that the three enzymes of the oxidative phase of the PPP have different susceptibilities to inactivation on exposure to different oxidants. In this review, we discuss existing knowledge on the role that these enzymes play in the metabolism of cells, and their susceptibility to oxidation and inactivation with special emphasis on NADPH production. Perspectives on achieving a better understanding of the molecular basis of the oxidation these enzymes within cellular environments are given.