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.
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ABSTRACT: Thioredoxins, glutaredoxins, and peroxiredoxins have been characterized as electron donors, guards of the intracellular redox state, and 'antioxidants'. Today, these redoxins are increasingly recognized for their specific role in redox signaling. Redoxin research is by no means 'old-fashioned'; on the contrary, the number of publications on the topic continues to increase exponentially. This review summarizes the almost 50 years of redoxin research, focusing primarily on recent data from vertebrates and mammals. The role of Trx family and related proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners of the redoxins. On basis of this analysis, the importance of the redoxins for human health is addressed in the second part of this review, i.e. their potential impact and functions in different cell types, metabolic and signaling pathways, and various pathological conditions.Antioxidants and Redox Signaling 11/2013; 19(13):1539-1605. · 7.19 Impact Factor
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ABSTRACT: Glutaredoxins (Grx) are small proteins, conserved throughout all the kingdoms of life, which are engaged in a wide variety of biological processes. According to the number of cysteines in their active site, Grx are classified as dithiolic or monothiolic (1-C-Grx). In most organisms, 1-C-Grx are implicated in iron-sulfur cluster (FeS) metabolism and utilize glutathione as cofactor. Trypanosomatids are parasitic protozoa of the order Kinetoplastida, which cause severe diseases in humans and domestic animals. These parasites exploit a unique thiol-dependent redox system based on bis(glutathionyl)spermidine (trypanothione) rather than on glutathione. Mitochondrial 1-C-Grx1 from trypanosomes differs from orthologues in several features including the use of trypanothione as ligand for FeS binding and the presence of a parasite-specific N-terminal extension. We have recently shown that 1-C-Grx1 from Trypanosoma brucei is indispensable for parasite survival in mouse, making this protein a potential drug target candidate against trypanosomiasis. However, structural information for the full-length form of 1-C-Grx1 is still lacking. Here, we report the NMR resonance assignment of the mature form of Tb1-C-Grx1 including an N-terminal tail, paving the way to disclose the role of this intrinsically disordered region in the protein function.Biomolecular NMR Assignments 05/2014; · 0.64 Impact Factor
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ABSTRACT: Lower eukaryotes like the kinetoplastid parasites are good models to study evolution of cellular pathways during steps to Eukaryogenesis. In this study, a kinetoplastid parasite Leishmania donovani was used to understand the process of mitochondrial translocation of a nuclear encoded mitochondrial protein, the mitochondrial tryparedoxin peroxidase (mTXNPx). We report the presence of an N-terminal cleavable mitochondrial targeting signal (MTS) validated through deletion and grafting experiments. We also establish a novel finding of calmodulin (CaM) binding to the MTS of mTXNPx through specific residues. Mutation of CaM binding residues keeping intact the residues involved in mitochondrial targeting and biochemical inhibition of CaM activity both in vitro and in vivo prevented mitochondrial translocation. Through reconstituted import assays, we demonstrate obstruction of mitochondrial translocation either in absence of CaM or Ca(2+) or in the presence of CaM inhibitors. We also demonstrate the prevention of temperature driven mTXNPx aggregation in the presence of CaM. These findings establish the idea that CaM is required for the transport of the protein to mitochondria through maintenance of translocation competence post translation.Molecular and cellular biology 09/2013; · 6.06 Impact Factor