Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

Abstract

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…The monothiol Grxs have been partially characterized, and we could show that 1-C-Grx1 is a mitochondrial protein that forms an iron-sulfur cluster (25,26). Despite the absence of a GR, trypanosomatids contain appreciable concentrations of free GSH (13).…”

“…Generation of Recombinant T. brucei Grx1 and Grx2-In the case of the T. brucei monothiol 1-C-Grx1, the starting Met proved not to be encoded by the first ATG following a stop codon (25). Therefore, the 5Ј-regions of the mRNAs were amplified from cDNA using gene-specific reverse primers in combination with a spliced leader primer.…”

Mono- and Dithiol Glutaredoxins in the Trypanothione-Based Redox Metabolism of Pathogenic Trypanosomes

“…Similarly to 2-C-Grxs, distinct isoforms of 1-C-Grxs are found in the cytosol, nucleus, mitochondria as well as plant chloroplast (reviewed in 86). Except for yeast Grx6 and Grx7 (59,60), most 1-C-Grxs lack or have negligible classical disulfide reductase activity (18,26,27,43,69,82). Instead, the capability to coordinate ISCs appears to be a common feature for 1-C-Grxs (16,40,42,43,54,67,89) with yeast Grx7 being, so far, the only known exception (59,60).…”

“…The parasite Grxs have a variety of distinctive biochemical, structural and biological features, and are functionally linked to the trypanothione system (12,16,27,56) (Table 1). These proteins are subject of this review.…”

“…Recombinant T. brucei 1-C-Grx1 exists as a noncovalent homodimer in both the apo-form and with bound iron-sulfur cluster, whereas the apo-forms of 1-C-Grx2 and 1-C-Grx3 are monomeric proteins (16,27). An N-terminal region of about *37 residues, exclusive of the kinetoplastid 1-C-Grx1s, has recently been identified to be responsible for dimerization of the T. brucei and T. cruzi proteins (55a).…”