NEWS &VIEWS Cloning, Expression, and Characterization of a Dithiol Glutaredoxin from Trypanosoma cruzi Vanina E. Marquez, 1 Diego G. Arias, 1 Claudia V. Piattoni, 1 Carlos Robello, 2 Alberto A. Iglesias, 1 and Sergio A. Guerrero 1 Abstract Glutaredoxins play an important role in cellular functionality. A putative dithiol glutaredoxin is encoded in the genome of Trypanosoma cruzi. We cloned the gene and obtained the recombinant protein, which behaved as a typical thioltransferase. Activity was variable and dependent on the nature of reducer or oxidant agent used, or both. Epimastigote extracts exhibited similar activity, suggesting the occurrence of the protein in the parasite. Results support a redox scenario in T. cruzi, with glutaredoxin being involved mainly in reduction of glutathione disulfide as well as in deglutathionylation of target proteins. Antioxid. Redox Signal. 12, 787–792. Introduction T rypanosoma cruzi is the causative agent of Chagas disease. As usual for aerobic organisms, the parasite is exposed to several reactive oxygen species (ROS): superoxide anions, hydrogen peroxide, and myeloperoxidase-derived products. These chemical species are generated during the host-defense reaction and also as by-products of the aerobic metabolism. The ability of T. cruzi to cope with such oxidative conditions appears oddly weak. Although trypanosomatids possess an iron-containing superoxide dismutase to scavenge phagocyte-derived superoxide anions (17), they lack catalase, and their glutathione peroxidase-like system exhibits low effi- ciency (10, 26). Whereas catalase and selenocysteine-containing glutathione peroxidases are the major hydroperoxide- metabolizing enzymes in host organisms (3, 21), in members of the family Trypanosomatidae, the peroxide metabolism involves mainly a glutathionyl derivative of spermidine, try- panothione [N 1 ,N 8 -bis (glutathionyl)-spermidine, T(SH) 2 ] (7, 14). A system linking three distinctive oxidoreductases is able to catalyze the T(SH) 2 -dependent hydroperoxide removal. These enzymes are trypanothione reductase (TR) (7, 14), a thioredoxin-related protein called tryparedoxin, tryparedoxin peroxidase (TXNPx), and peroxiredoxin-type proteins (or glutathione peroxidase type) (10, 28). Additionally, a classic thioredoxin was described and characterized in T. cruzi (24), but neither glutathione reductase nor thioredoxin reductase is found in these microorganisms (11, 23). Glutaredoxins (Grxs) are ubiquitous oxidoreductases be- longing to the thioredoxin family of proteins (18). They are classified as monothiolic or dithiolic Grxs after the respective occurrence of the conserved motifs CGFS or CXYC into the redox active site. Dithiolic Grxs reduce glutathionylated compounds or intramolecular disulfide bonds by a monothiol or a dithiol mechanism (6). Among the many functions pro- posed for dithiolic Grxs, they were first identified as electron donors of ribonucleotide reductase in the generation of de- oxyribonucleotides (8, 18). They also were characterized as substituting thioredoxin as reducer of phosphoadenylylsul- fate reductase in bacteria, plants, and yeasts (18). Further- more, Grxs can function as general glutathione-dependent protein disulfide oxidoreductases (6), catalyzing the reduction of glutathionylated compounds (5), as well as glutathionyla- tion and deglutathionylation of specific proteins (4). The present work reports the molecular cloning, expression, and purification of a dithiolic Grx from T. cruzi and functional studies supporting the protein as a key component of the redox metabolic scenario in the parasite. This is the first time that this kind of Grx has been characterized in trypanoso- matids. Molecular Cloning and Heterologous Expression of T. cruzi Dithiol Grx In the T. cruzi CL Brener database (http:==tcruzidb.org), we identified two putative grx genes (Tc00.1047053506475.116 and Tc00.1047053511431.40) having 97.8% identity between them. Both genes code for an identical protein at the level of an amino acid sequence; which shows 22 to 30% identity with other reported Grxs. Sequence alignment detailed in Fig. 1 1 Instituto de Agrobiotecnologı ´a del Litoral, UNL-CONICET, Ciudad Universitaria-Paraje ‘‘El Pozo,’’ Santa Fe, Argentina. 2 Departamento de Bioquı ´mica, Facultad de Medicina, Universidad de la Repu ´ blica and Unidad de Biologı ´a Molecular, Instituto Pasteur Montevideo, Montevideo, Uruguay. ANTIOXIDANTS & REDOX SIGNALING Volume 12, Number 6, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=ars.2009.2907 787