title: Functional characterization of peroxiredoxins from the human protozoan parasite Giardia intestinalis.
abstract: The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O2 and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O2, NO and superoxide anion (O2 (-•)) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H2O2 and peroxynitrite (ONOO(-)). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H2O2 and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO(-) rapidly (k = 4×10(5) M(-1) s(-1) and 2×10(5) M(-1) s(-1) at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O2 for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.
authors.affiliation: CNR Institute of Molecular Biology and Pathology, Rome, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Mastronicola
authors.affiliation: Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.
authors.forename: Micol
authors.initials: M
authors.lastname: Falabella
authors.affiliation: Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Testa
authors.affiliation: L.Spallanzani National Institute for Infectious Diseases, IRCCS, Rome, Italy.
authors.forename: Leopoldo Paolo
authors.initials: LP
authors.lastname: Pucillo
authors.affiliation: Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Teixeira
authors.affiliation: CNR Institute of Molecular Biology and Pathology, Rome, Italy ; Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Sarti
authors.affiliation: Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Lígia M
authors.initials: LM
authors.lastname: Saraiva
authors.affiliation: CNR Institute of Molecular Biology and Pathology, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Giuffrè
mesh_terms: D000818:Animals; D001555:Benzene Derivatives; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D054464:Peroxiredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020122:tert-Butylhydroperoxide
chemical_list: D001555:Benzene Derivatives; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D009249:NADP; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; C007164:cumene hydroperoxide