Authors
R B Currier1; A Leroux2; K Ulrich3; N Dirdjaja1; R L Krauth-Siegel1; 1 Biochemie-Zentrum der Universität Heidelberg, Germany; 2 Biomedicine Research Institute of Buenos Aires, Argentina; 3 University of Michigan, United States Discussion
Trypanosomatids possess a unique redox metabolism based on the trypanothione/trypanothione reductase system. The majority of reactions are mediated by tryparedoxin, the main oxidoreductase found in the parasite cytosol, however maintenance of the thiol-redox homeostasis in the mitochondrion is not yet fully understood.
The Trypanosoma genome encodes two thioredoxin-type proteins, Trx1 and Trx2. Previous studies showed that Trx1 functions as a conventional oxidoreductase, whereas the role of Trx2, a protein unique to Kinetoplastids, is currently unknown. As thioredoxin reductases have not been identified in Kinetoplastid organisms, the trypanothione system is the only known source of reducing equivalents for any type of oxidoreductases.
Here, we report that Trx2 is a mitochondrial protein and is essential for proliferation of both bloodstream and procyclic T. brucei parasites. Depletion of Trx2 in procyclic cells causes a proliferation defect which is overcome after approximately 8 days of RNAi induction. Remarkably, this is not due to reappearance of the protein suggesting that this occurs via a compensatory mechanism involving the over-expression of a protein with a similar function. Interestingly, this mechanism is accelerated following heat stress.
Conditional knock-out cell lines expressing a mutant Trx2 which lacks all five cysteine residues are viable and proliferative, and show no differences in response to heat shock or oxidative stress compared to wild type cells. We also demonstrate that recombinant Trx2 as well as the 5-cysteine mutant protein both inhibit, instead of accelerate, protein precipitation in the insulin reduction assay. These findings suggest that the primary role of T. brucei Trx2 may be a chaperone function that is independent of its thiol redox activity. 
