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Abstract

Glutaredoxins (Grxs) are small ubiquitous oxidoreductases. Trypanosoma brucei, the causative of African sleeping sickness, contains two dithiol glutaredoxins. Previous studies had shown that RNA interference against Grx2 resulted in a proliferation defect of the procyclic insect form whilst depletion of Grx1 did not show any phenotype in both the mammalian bloodstream and procyclic cells. The aim of my thesis was to investigate the physiological roles of Grx1 and Grx2 in more detail. For this purpose, I generated bloodstream cell lines lacking both alleles of Grx1 or Grx2. Under normal culture conditions, the mutant cells did not show any growth defect, however, remarkably, displayed an improved proliferation compared to wildtype cells when cultured at elevated temperature. Immunofluorescence microscopy of DAPI-stained cells showed in the wildtype population a significantly higher proportion of parasites with at least two kinetoplasts and two nuclei when compared to the knockout cell lines. The two glutaredoxins cannot substitute for each other. In bloodstream cells there was no upregulation of either protein when the other one was absent. Treatment of recombinant Grx1 with GSH/diamide resulted in the glutathionylation of its non-active site third cysteine accompanied by a three-fold decrease in activity to reduce glutathione-mixed disulfides. Procyclic Grx1-deficient cells could also be obtained. The protein proved to be dispensable even in the presence of different stresses. However, in the case of Grx2, the second allele could only be replaced in cells possessing an inducible ectopic copy of either wildtype or C34S Grx2, but not the C31S/C34S mutant. Both types of cell lines displayed leaky expression of the ectopic copy when the second grx2 allele was knocked out, suggesting an essential role for this protein in procyclic cells as the system was tightly controlled in parasites still having one grx2 allele. These results show that Grx2 has a redox function in procyclic cells requiring at least the N-terminal active site cysteine. In vitro assays with wildtype and C34S Grx2 suggested that the protein is mainly involved in deglutathionylation reactions. I also showed that Grx1 and Grx2 are fully in the dithiol state in bloodstream and procyclic cells, respectively, and return to their reduced states within 5 min after the removal of an oxidative stress.