The products were pHD1700/and pHD1700/were harvested in the logarithmic growth phase. parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under Rabbit polyclonal to AMN1 these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component. provide a kinetic barrier that prevents the reduction of target proteins by glutathione (GSH) . A unique feature of dithiol Grxs is their ability to catalyze redox reactions using only the first cysteine (monothiol reactions). Generally, the functions of Grxs are closely linked to the GSH system since (i) their reduced form is regenerated by thiol/disulfide exchange of the oxidized Salvianolic acid C protein with GSH, where the GSSG formed is then reduced by glutathione reductase, and (ii) they catalyze with high efficiency and selectivity the reversible S-glutathionylation of proteins. The latter mechanism may be employed to protect reactive cysteine residues in distinct proteins from irreversible over-oxidation as well as for redox signaling pathways that could mediate critical cellular functions like proliferation and apoptosis , , , . Trypanosomatids, such as the causative agent of African sleeping sickness and Nagana cattle disease, lack glutathione reductases and thioredoxin reductases and their thiol metabolism is based on the Salvianolic acid C low molecular mass dithiol trypanothione Salvianolic acid C [bis(glutathionyl)spermidine, T(SH)2] and trypanothione reductase (for reviews see , , ). T(SH)2 is synthesized from two molecules of GSH that are covalently linked by spermidine with glutathionylspermidine (Gsp) as intermediate , . The T(SH)2 system is involved in the synthesis of DNA precursors as well as the detoxification of hydroperoxides. The reactions are mediated by tryparedoxin (Tpx). This essential and parasite-specific oxidoreductase is a distant member of the thioredoxin-type protein family and fulfils many of the functions known to be catalyzed by thioredoxins and/or Grxs in other organisms , . Despite the absence of a classical glutathione system, trypanosomatids contain appreciable concentrations of free GSH as well as a repertoire of distinct Grxs , . Recently we showed that as response to exogenous and endogenous oxidative stresses, the mammalian bloodstream (BS) form of can undergo protein S-glutathionylation and S-trypanothionylation . The genome encodes genes for three monothiol Grxs as well as two dithiol Grxs (Grx1 and Grx2) . Grx1 represents a canonical dithiol Grx whereas Grx2 has sequence features exclusively found in trypanosomatid organisms . In gene. The protein has an overall sequence identity of 80% with Grx2 and is located in the cytosol . The catalytic properties of recombinant Grx1 and Grx2 as well as Grx have been studied in some detail , , . The reduced form of the proteins with the active site cysteines (Cys31 and Cys34 in Grx2) in the thiol state is regenerated from the intramolecular disulfide by spontaneous thiol/disulfide exchange with T(SH)2, reactions that are at least three orders of magnitude faster compared to those with GSH , . The trypanosomal Grxs accelerate the reduction of GSSG by T(SH)2 which again reflects their close link with the trypanothione metabolism. Both Grxs and Grx catalyze the reduction of the mixed disulfide between GSH and either 2-mercaptoethanol or cysteine residues of various model proteins, a reaction that is not taken over, at least to a physiological competent degree, by Tpx , , . Indeed, the cytosolic Grx1 has been shown to contribute to about 50% of the deglutathionylation capacity of infective and confers resistance against oxidative damage and promotes parasite growth while in non-infective parasites it induces apoptosis . Here we investigated the molecular and biological details of the overall contribution of the Grx-dependent metabolism for parasite survival in an animal host as well as of the indispensability of Grx2 for PC trypanosomes. We show that Grx2 specifically localizes to the IMS of the mitochondrion and that its biological functions require the presence of both (in BS cells) or only the first (in PC cells) of the active site cysteine residues. Grx2 was dispensable for infective trypanosomes but, as observed for Grx1 KO cells, its absence increased the thermo-tolerance of BS cells. Thus, from a therapeutic point of view, the parasite Grxs can be ruled out as putative.