Browsing by Author "Leinisch, Fabian"

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    Aggregation of alpha- and beta- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation
    (2018) Fuentes Lemus, Eduardo Felipe; Silva, Eduardo; Barrias, Pablo; Aspee, Alexis; Escobar Álvarez, Elizabeth; Lorentzen, Lasse G.; Carroll, Luke; Leinisch, Fabian; Davies, Michael J.; López Alarcón, Camilo Ignacio
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    Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan
    (2019) Fuentes Lemus, Eduardo Felipe; Mariotti, Michele; Hägglund, Per; Leinisch, Fabian; Fierro Huerta, Angélica; Silva, Eduardo; López Alarcón, Camilo Ignacio; Davies, Michael J.
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    Oxidation of lysozyme induced by peroxyl radicals involves amino acid modifications, loss of activity, and formation of specific crosslinks
    (2021) Fuentes Lemus, Eduardo Felipe; Mariotti, Michele; Hägglund, Per; Leinisch, Fabian; Fierro Huerta, Angélica; Silva Stevens, Eduardo Andrés; Davies, Michael J.; López Alarcón, Camilo Ignacio
    The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO?). We hypothesized that exposure to ROO? would generate specific modifications and inter- and intraprotein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2?-azobis(2-methylpropionamidine) dihydrochloride) as a ROO? source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 ?C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO?). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLCMS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.
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    Peroxyl radical- and photo-oxidation of glucose 6-phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues
    (2017) Leinisch, Fabian; Mariotti, Michele; Rykaer, Martin; López Alarcón, Camilo Ignacio; Davies, Michael J.; Hagglund, Per
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    Role 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 Ignacio
    The 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.
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    The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function.
    (2017) Escobar Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela; Monasterio, Octavio; Lorentzen, Lasse G.; Silva, Eduardo; Davies, Michael J.; López Alarcón, Camilo Ignacio