María Dolores Piñeyro1, Diego Arias2, Alejandro Ricciardi3, Carlos Robello1, Adriana Parodi-Talice4. 1. Unidad de Biología Molecular, Institut Pasteur Montevideo, Montevideo, Uruguay; Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2. Laboratorio de enzimología molecular, Instituto de agrobiotecnología del litoral, UNL-CONICET, Santa Fe, Argentina; Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina. 3. Laboratorio de Biofármacos: Control de Calidad & Desarrollo, Institut Pasteur Montevideo, Montevideo, Uruguay. 4. Unidad de Biología Molecular, Institut Pasteur Montevideo, Montevideo, Uruguay; Sección Genética, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Electronic address: apartal@fcien.edu.uy.
Abstract
BACKGROUND: Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor. METHODS: Four residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined. RESULTS: In this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates. CONCLUSIONS: c-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative CP and CR role. GENERAL SIGNIFICANCE: This study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx.
BACKGROUND:Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor. METHODS: Four residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined. RESULTS: In this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates. CONCLUSIONS:c-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative CP and CR role. GENERAL SIGNIFICANCE: This study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx.
Authors: Lucía López; María Laura Chiribao; Magalí C Girard; Karina A Gómez; Paula Carasi; Marisa Fernandez; Yolanda Hernandez; Carlos Robello; Teresa Freire; María Dolores Piñeyro Journal: Immunology Date: 2021-01-25 Impact factor: 7.397