title: The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.
abstract: Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. shane.wilkinson@lshtm.ac.uk
authors.forename: Shane R
authors.initials: SR
authors.lastname: Wilkinson
authors.forename: David J
authors.initials: DJ
authors.lastname: Meyer
authors.forename: Martin C
authors.initials: MC
authors.lastname: Taylor
authors.forename: Elizabeth V
authors.initials: EV
authors.lastname: Bromley
authors.forename: Michael A
authors.initials: MA
authors.lastname: Miles
authors.forename: John M
authors.initials: JM
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014349:Trypanosoma cruzi / Q000201:enzymology*; D000094765:Glutathione Peroxidase GPX1
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; C114110:tryparedoxin; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D012313:RNA; D004247:DNA; C044809:trypanothione; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005978:Glutathione; D010100:Oxygen; D013095:Spermidine; D000094765:Glutathione Peroxidase GPX1