1H, 13C and 15N resonance assignment of the cytosolic dithiol glutaredoxin 1 from the pathogen Trypanosoma brucei

Abstract

Trypanosomatids are parasites responsible for several tropical and subtropical diseases, such as Chaga's disease, sleeping sickness and Leishmaniasis. In contrast to the mammalian host, the thiol-redox metabolism of these pathogens depends on trypanothione [bis-glutathionylspermidine, T(SH)2] instead of glutathione (GSH) providing a set of lineage-specific proteins as drug target candidates. Glutaredoxins (Grx) are ubiquitous small thiol-disulfide oxidoreductases that belong to the thioredoxin-fold family. They play a central role in redox homeostasis and iron sulfur-cluster biogenesis. Each species, including trypanosomes, possesses its own set of isoforms distributed in different subcellular compartments. The genome of trypanosomatids encodes for two class I (dithiolic) Grxs named 2-C-Grx1 and 2-C-Grx2. Both proteins were shown to efficiently reduce different disulfides at the expenses of T(SH)2 using a mechanism that involves the two cysteines in the active site. Moreover, the cytosolic Trypanosoma brucei 2-C-Grx1 but not the mitochondrial 2-C-Grx2 was able to coordinate an iron-sulfur cluster with T(SH)2 or GSH as ligand. As a first step to unravel the structural basis for the specificity observed in the trypanosomal glutaredoxins, we present here the NMR resonance assignment of 2-C-Grx1 from the parasite T. brucei brucei.

Nuclear factor‐kappa B (NF‐κB) in pathophysiology of Parkinson disease: Diverse patterns and mechanisms contributing to neurodegeneration

“…LPS‐induced activation of NF‐κB, through NF‐κB /Nurr1 axis, culminates in RelA binding to the promoter region of Glutaredoxin‐1 (Grx1), the common sulfhydryl disulfide oxidoreductase in microglia (Stefani et al., 2016; Upadhyaya et al., 2015), and thereby through decreased S‐glutathionylation of IKKβ, results in accelerated NF‐κB activation and cytokine production. Thus, a feed‐forward loop between NF‐κB activation and Grx1, which results in neuroinflammation, is formed (Aesif et al., 2011; Gorelenkova Miller et al., 2016).…”

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites

“…5). Interestingly, although the assignment of the oxidized protein is not available, from the assignment of Grx1(SH) 2 (31) it is evident that the most prominent shifts occurred at residues that partake in the GSH-binding cleft (Fig. 5).…”

“…Due to the cloning strategy, all tag-free versions of Grx1 contain an N-terminal GAME sequence instead of Met 1 yielding a calculated molecular weight of 10,833.49. 15 N uniformly labeled Grx1 for NMR was prepared as previously reported (31) by growing E. coli BL21(DE3)-transformed cells in M9 minimal medium containing 1 g/liter of 15 NH 4 Cl as the only nitrogen source. The labeled protein was expressed and purified as described above and the buffer exchanged to 50 mM sodium phosphate, pH 7.2, 150 mM NaCl (buffer C) with 10 mM DTT in H 2 O/D 2 O (95:5%, v/v) and the protein concentrated by ultrafiltration.…”