Hugo, M.; Martínez, A.; Trujillo, M.; Estrada, D.; Mastrogiovanni, M.; Linares, E.; Augusto, O.; Issoglio, F.; Zeida, A.; Estrín, D.A.; Heijnen, H.F.G.; Piacenza, L.; Radi, R."Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)" (2017) Proceedings of the National Academy of Sciences of the United States of America. 114(8):E1326-E1335
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The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1·s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1·s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium. © 2017, National Academy of Sciences. All rights reserved.


Documento: Artículo
Título:Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)
Autor:Hugo, M.; Martínez, A.; Trujillo, M.; Estrada, D.; Mastrogiovanni, M.; Linares, E.; Augusto, O.; Issoglio, F.; Zeida, A.; Estrín, D.A.; Heijnen, H.F.G.; Piacenza, L.; Radi, R.
Filiación:Departamento de Bioquímica, Facultad de Medicina, Universidad de La República, Montevideo, 11800, Uruguay
Center for Free Radical and Biomedical Research, Universidad de La República, Montevideo, 11800, Uruguay
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Sao Paulo, 05508-000, Brazil
Departamento de Química Inorgánica, Analítica y Química-Física, Instituto de Química Física de Materiales, Medio Ambiente y Energía (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EHA, Argentina
Department of Clinical Chemistry and Hematology, Cell Microscopy Core, University Medical Center, Utrecht, 3584CX, Netherlands
Department of Molecular Toxicology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal, 14558, Germany
Palabras clave:Heme peroxidase; Kinetics; Oxidants; Trypanosoma cruzi; Virulence; ascorbate peroxidase; cytochrome c peroxidase; heme; hydrogen peroxide; phenylalanine; cytochrome c; peroxidase; tryptophan; absorption spectroscopy; amastigote; animal cell; animal experiment; Article; cell kinetics; cell membrane; cellular distribution; controlled study; electron spin resonance; electron transport; enzyme activity; enzyme mechanism; enzyme substrate; host parasite interaction; mass spectrometry; mitochondrial membrane; molecular dynamics; mouse; nonhuman; parasite virulence; priority journal; protein expression; spin trapping; Trypanosoma cruzi; animal; Bagg albino mouse; C57BL mouse; Chagas disease; female; kinetics; male; metabolism; oxidation reduction reaction; parasite; parasitology; pathogenicity; physiology; procedures; site directed mutagenesis; Trypanosoma cruzi; virulence; Animals; Chagas Disease; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Electron Transport; Female; Heme; Hydrogen Peroxide; Kinetics; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Oxidation-Reduction; Parasites; Peroxidase; Phenylalanine; Trypanosoma cruzi; Tryptophan; Virulence
Página de inicio:E1326
Página de fin:E1335
Título revista:Proceedings of the National Academy of Sciences of the United States of America
Título revista abreviado:Proc. Natl. Acad. Sci. U. S. A.
CAS:ascorbate peroxidase, 72906-87-7; cytochrome c peroxidase, 9029-53-2; heme, 14875-96-8; hydrogen peroxide, 7722-84-1; phenylalanine, 3617-44-5, 63-91-2; cytochrome c, 9007-43-6, 9064-84-0; peroxidase, 9003-99-0; tryptophan, 6912-86-3, 73-22-3; Cytochrome c Group; Heme; Hydrogen Peroxide; Peroxidase; Phenylalanine; Tryptophan


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---------- APA ----------
Hugo, M., Martínez, A., Trujillo, M., Estrada, D., Mastrogiovanni, M., Linares, E., Augusto, O.,..., Radi, R. (2017) . Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP). Proceedings of the National Academy of Sciences of the United States of America, 114(8), E1326-E1335.
---------- CHICAGO ----------
Hugo, M., Martínez, A., Trujillo, M., Estrada, D., Mastrogiovanni, M., Linares, E., et al. "Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)" . Proceedings of the National Academy of Sciences of the United States of America 114, no. 8 (2017) : E1326-E1335.
---------- MLA ----------
Hugo, M., Martínez, A., Trujillo, M., Estrada, D., Mastrogiovanni, M., Linares, E., et al. "Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)" . Proceedings of the National Academy of Sciences of the United States of America, vol. 114, no. 8, 2017, pp. E1326-E1335.
---------- VANCOUVER ----------
Hugo, M., Martínez, A., Trujillo, M., Estrada, D., Mastrogiovanni, M., Linares, E., et al. Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP). Proc. Natl. Acad. Sci. U. S. A. 2017;114(8):E1326-E1335.