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. 2016 Jan 27;24(4):205-216.
doi: 10.1089/ars.2015.6377. Epub 2015 Oct 27.

A New Class of Thioredoxin-Related Protein Able to Bind Iron-Sulfur Clusters

Affiliations

A New Class of Thioredoxin-Related Protein Able to Bind Iron-Sulfur Clusters

Hugo Bisio et al. Antioxid Redox Signal. .

Abstract

Aims: Members of the thioredoxin (Trx) protein family participate mainly in redox pathways and have not been associated with Fe/S binding, in contrast to some closely related glutaredoxins (Grxs). Cestode parasites possess an unusual diversity of Trxs and Trx-related proteins with unexplored functions. In this study, we addressed the biochemical characterization of a new class of Trx-related protein (IsTRP) and a classical monothiol Grx (EgGrx5) from the human pathogen Echinococcus granulosus.

Results: The dimeric form of IsTRP coordinates Fe2S2 in a glutathione-independent manner; instead, Fe/S binding relies on the CXXC motif conserved among Trxs. This novel binding mechanism allows holo-IsTRP to be highly resistant to oxidation. IsTRP lacks canonical reductase activities. Mitochondrially targeted IsTRP aids growth of a Grx5 null yeast strain. Similar complementation assays performed with EgGrx5 revealed functional conservation for class II Grxs, despite the presence of nonconserved structural elements. IsTRP is a cestode lineage-specific protein highly expressed in the gravid adult worm, which releases the infective stage critical for dissemination.

Innovation: IsTRP is the first member from the Trx family to be reported to bind Fe/S. We disclose a novel mechanism of Fe/S coordination within the Trx folding unit, which renders the cluster highly resistant to oxidation-mediated disassembly.

Conclusion: We demonstrate that IsTRP defines a new protein family within the Trx superfamily, confirm the conservation of function for class II Grx from nonphylogenetically related species, and highlight the versatility of the Trx folding unit to acquire Fe/S binding as a recurrent emergent function. Antioxid. Redox Signal. 00, 000-000.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
IsTRP and Grx5 alignment and phylogenetic analysis. (A) Simplified unrooted neighbor-joining phylogeny of some Trx superfamily members (Trxs, thioredoxins; Trx domain of PDI, thioredoxin domain of protein disulfide isomerases; IsTRP, Iron–sulfur thioredoxin-related protein; Grxs, glutaredoxins; Prdxs, peroxiredoxins; Trx-like Frd, thioredoxin-like ferredoxin). Bootstrap values were calculated using 500 iterations. The members of the Trx fold that can coordinate Fe/S are shown in gray boxes (for IsTRP, the evidence stems from this work). Sequences specific to the Cyclophyllidea order within the class Cestoda are pointed with a black circle on the branches. (B, C) Protein sequence alignment of IsTRP and Grx5 ortholog proteins, respectively. Cysteine residues are highlighted in black. The residues of the insertion of tapeworm Grx5 are shown in bold. PSS elements (highlighted in gray) are represented with (H) when it is α-helix, (S) when it is β-sheet, and (C) when it is coiled. Ts. Taenia solium (IsTRP KR152308). Ta. Taenia asiatica (IsTRP KR152310). Eg. Echinococcus granulosus (IsTRP CDS20645, Grx5 EUB62482). Tt. Taenia taeniaeformis (IsTRP KR152311). Em. Echinococcus multilocularis (IsTRP CDJ06281). Hm. Hymenolepis microstoma (IsTRP KR152312). Hn. Hymenolepis nana (IsTRP KR152314). Hd. Hymenolepis diminuta (IsTRP KR152313). Sm. Schistosoma mansoni (Grx5 CCD60523). Hs. Homo sapiens (Grx5 AAH23528). Schm. Schmidtea mediterranea. Sc. Saccharomyces cerevisiae (Grx5 3GX8). Ec. Escherichia coli (Grx5 2WCI). Fe/S, iron–sulfur cluster; PSS, predicted secondary structure.
<b>FIG. 2.</b>
FIG. 2.
SEC and spectroscopic analysis of E. granulosus IsTRP and Grx5. Size exclusion chromatography analysis of IsTRP and Grx5 is shown in (A, C), respectively, and was performed on a Superdex 75 10/300 GL column. Absorbance at 280, 320, and 420 nm is shown for EDTA-treated (ET) and nontreated (NT) samples. The UV-visible spectra and SDS-PAGE of fractions F1 (black) and F2 (gray) of the untreated samples are shown for IsTRP and Grx5 in (B, D), respectively. SDS-PAGE of recombinant proteins before (left lane) and after (right lane) gel filtration is shown as an inset. (E, F) Size exclusion chromatography of Fe/S reconstitution mixture for tag-free IsTRP and Grx5, respectively. Absorbance of the proteins AR and BR is shown. The spectra of the reconstituted proteins are shown in an inset. AR, after reconstitution; BR, before reconstitution; ET, EDTA-treated; NT, nontreated; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
<b>FIG. 3.</b>
FIG. 3.
IsTRP and Grx5 Fe/S-binding properties. (A, B) Kinetics for the disassembly of holo-IsTRP and holo-Grx5 (NT) and upon treatment (T) with 10 mM of H2O2 or 3 mM of GSH. The loss of the Fe/S was recorded by following the decrease in absorbance at 320 nm. Error bars correspond to standard deviations of three replicates. (C, D) The Fe/S coordination ability of wild-type, Cys38Ser, Cys37Ser, and Cys34Ser mutants was analyzed by measuring the absorbance at 320 nm of the copurified holoproteins. Protein concentration was between 300 and 200 μM. A representative UV-visible spectrum (normalized to protein concentration) of each protein species is shown (C) and the corresponding 320/280 nm ratio relative to the binding of the wild-type protein is shown (D). GSH, glutathione.
<b>FIG. 4.</b>
FIG. 4.
Activities of IsTRP and Grx5. (A) Insulin reduction by dithiothreitol. Twenty micromolars of IsTRP and Grx5; and 2 μM of a Trx from E. granulosus. The absorbance at 650 nm is plotted against time. (B) HED activity assay. The capacity of IsTRP, Grx5, and a Grx from E. granulosus to deglutathionylate was evaluated. NADPH oxidation was followed by measuring absorbance at 340 nm. The full time courses obtained are shown. (C) Reduction of 20 μM wild-type IsTRP (black) and mutants Cys to Ser by the TGRC31S (a TGR mutant that conserves full TR activity, but lacks gutathione reductase activity). One hundred twenty-five nanomolars of TGRC31S was used for the assay. Representative full time courses of NADPH oxidation are shown. TGR, thioredoxin glutathione reductase; TR, Thioredoxin reductase.
<b>FIG. 5.</b>
FIG. 5.
Rescue of the ΔGrx5 mutant defects by Grx5 and IsTRP. (A) Growth plates after a 3-day incubation at 30°C on YPD. From left to right, 1:5 serial dilutions of exponential cultures were spotted. (B) Growth on YPD or YPG plates after 3 days at 30°C. S. cerevisiae Grx5 (ScGrx5), E. granulosus (EgGrx5), E. granulosus IsTRP (IsTRP), and null mutant (ΔGrx5) are shown. (C) Ratio between the Fe/S-containing enzyme aconitase and the non-Fe/S-containing enzyme malate dehydrogenase activities in exponential cultures at 30°C in YPGal medium. Values were normalized with respect to strain MML240 that expresses yeast Grx5-HA. The values are the mean of three experiments with standard deviations. Statistical analysis was performed using one-way ANOVA (*p < 0.05).

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