Biology subjects

Tarrago, L.

Publications and source records attributed to Tarrago, L..

5 recordsLinked to original sources

bioRxiv2025

Quantitative mapping of methionine sensitivity to oxidation in the copper-bound PcuC chaperone

Copper is typically coordinated by histidine, cysteine, or methionine in proteins, and these residues are particularly sensitive to oxidation. However, it remains unclear whether copper-coordinating residues are more prone to oxidation than non-coordinating ones, and how their susceptibility changes between the apo and copper-bound states. The copper chaperone PcuC, important for cytochrome c oxidase assembly in bacteria, contains a canonical binding site composed of two histidines and two methionines (H51xnM63x22H86xM88), as well as a disordered C-terminal extension enriched in methionine and histidine. To quantify methionine oxidation sensitivity in both apo- and Cu-bound PcuC, we used a methionine-specific oxaziridine probe combined with mass spectrometry and compared labeling patterns to those generated by 18O-labeled hydrogen peroxide. We show that methionine residues display distinct oxidation sensitivities in the apoprotein, and that the oxaziridine reacts similarly to H218O2. Importantly, this probe enables quantification of methionine oxidation independently of hydroxyl radicals generated by copper-driven Fenton chemistry, which lacks residue specificity. In the copper-bound form, Cu binding strongly alters methionine reactivity, with a marked increase in oxidation of the coordinating Met63 and Met88. Structural analysis revealed that two copper ions occupy the canonical site, while the C-terminal extension does not contribute to coordination. Comparison of structural features and oxidation values showed that methionine sensitivity correlates with solvent exposure in the folded domain, but with local positive charge in the disordered region. These findings demonstrate that copper coordination modulates methionine oxidation, and that oxaziridine-based probes provide powerful tools for mapping oxidation sensitivity in (metallo)proteins.

biochemistry↗

bioRxiv2025

Global mapping of thioredoxin interacting proteins in Neurospora crassa

Thioredoxins (Trx) are essential thiol-oxidoreductases that regulate redox homeostasis by reducing oxidized cysteines in a wide range of target proteins. However, the Trx system and other redox regulation mechanisms remain poorly characterized in saprotrophic filamentous fungi. Here, we identified the components of the Neurospora crassa Trx system and uncovered potential redox-regulated proteins using Trx affinity chromatography. Genome search identified three Trx and a single thioredoxin reductase that we named TRX1, TRX2 TRX3 and TRR. Notably, TRX1 carries a C-terminal disordered extension of unknown function, conserved in two ascomycete taxa (Leotiomycetes and Sordariomycetes). Using recombinant cysteine-to-serine mutants of each Trx, we performed affinity chromatography and identified 1,998 proteins - approximately 19% of the N. crassa proteome. To rank the putative Trx targets, we applied a fold enrichment metric, comparing protein abundance before and after affinity chromatography. The average fold enrichment was four, with values reaching up to 117 for the most enriched protein, a DEAD/DEAH box helicase. Among the top-enriched proteins, we identified homologs of known human and plant Trx targets, like peroxiredoxins, as well as 93 transcription factors and 38 kinases. Additional potential Trx targets encompass, ubiquitination-related enzymes, Fe-S cluster assembly proteins, phospholipases, exonucleases, and chitin synthases. Moreover, components of multiprotein complexes were co-purified, reflecting both direct Trx interactions and indirect co-association. Overall, this study provides a global map of potential redox regulated proteins and Trx targets in N. crassa, laying the ground for future investigations into redox signaling in filamentous fungi.

microbiology↗

bioRxiv2023

Methionine oxidation of Carbohydrate-Active enZymes during white-rot wood decay

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well-documented, the impact of ROS on the oxidation of the secreted proteins remains unclear and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H 18O with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes. ImportanceThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot model Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

microbiology↗

bioRxiv2021

Distribution of methionine sulfoxide reductases in fungi and conservation of the free-methionine-R-sulfoxide reductase in multicellular eukaryotes

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi. HighlightsO_LIFree and protein-bound methionine can be oxidized into methionine sulfoxide (MetO). C_LIO_LIMethionine sulfoxide reductases (Msr) reduce MetO in most organisms. C_LIO_LISequence characterization and phylogenomics revealed strong conservation of Msr in fungi. C_LIO_LIfRMsr is widely conserved in unicellular and multicellular fungi. C_LIO_LISome msr genes were acquired from bacteria via horizontal gene transfers. C_LI

genomics↗

bioRxiv2018

The Rhodobacter sphaeroides methionine sulfoxide reductase MsrP can reduce R- and S- diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown as absolutely stereospecific for the reduction of S- and R-diastereomer, respectively. Recently, the molybdenum-containing protein MsrP, conserved in all gram-negative bacteria, was shown to be able to reduce MetO of periplasmic proteins without apparent stereospecificity in Escherichia coli. Here, we describe the substrate specificity of the Rhodobacter sphaeroides MsrP. Proteomics analysis coupled to enzymology approaches indicate that it reduces a broad spectrum of periplasmic oxidized proteins. Moreover, using model proteins, we demonstrated that RsMsrP preferentially reduces unfolded oxidized proteins and we confirmed that this enzyme, like its E. coli homolog, can reduce both R- and S-diastereomers of MetO with similar efficiency.

biochemistry↗