Browsing by Author "Reyes Valenzuela, Juan Sebastián"
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- ItemCrowding modulates the glycation of plasma proteins: In vitro analysis of structural modifications to albumin and transferrin and identification of sites of modification(2022) Fuentes Lemus, Eduardo Felipe; Reyes Valenzuela, Juan Sebastián; López Alarcón, Camilo Ignacio; Davies, Michael J.Protein modification occurs in biological milieus that are characterized by high concentrations of (macro)mol-ecules (i.e. heterogeneous and packed environments). Recent data indicate that crowding can modulate the extent and rate of protein oxidation, however its effect on other post-translational modifications remains to be explored. In this work we hypothesized that crowding would affect the glycation of plasma proteins. Physiologically-relevant concentrations of albumin (35 mg mL-1) and transferrin (2 mg mL-1) were incubated with methylglyoxal and glyoxal (5 mu M-5 mM), two alpha-oxoaldehyde metabolites that are elevated in the plasma of people with diabetes. Crowding was induced by adding dextran or ficoll polymers. Electrophoresis, electron microscopy, fluorescence spectroscopy and mass spectrometry were employed to investigate the structural consequences of glycation under crowded conditions. Our data demonstrate that crowding modulates the extent of formation of transferrin cross-links, and also the modification pathways in both albumin and transferrin. Arginine was the most susceptible residue to modification, with lysine and cysteine also affected. Loss of 0.48 and 7.28 arginine residues per protein molecule were determined on incubation with 500 mu M methylglyoxal for albumin and transferrin, respectively. Crowding did not influence the extent of loss of arginine and lysine for either protein, but the sites of modification, detected by LC-MS, were different between dilute and crowded conditions. These data confirm the relevance of studying modification processes under conditions that closely mimic biological milieus. These data unveil additional factors that influence the pattern and extent of protein modification, and their structural consequences, in biological systems.
- ItemEffect of macromolecular crowding on protein oxidation: Consequences on the rate, extent and oxidation pathways(2021) Fuentes Lemus, Eduardo Felipe; Reyes Valenzuela, Juan Sebastián; Gamon, Luke F.; López Alarcón, Camilo Ignacio; Davies, Michael J.Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 μM min−1 without and with dextran (60 mg mL−1) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations.
- 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.
- ItemPeroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity(ELSEVIER SCIENCE INC, 2023) Reyes Valenzuela, Juan Sebastián; Fuentes Lemus, Eduardo Felipe; Romero, Jefferson; Arenas, Felipe; Fierro Huerta, Angelica María; Davies, Michael J.; Lopez Alarcon Camilo Ignacio6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO center dot, from AAPH (2,2 '-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100 mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO center dot induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO center dot elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO center dot and when compared to other PPP enzymes.
- 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.