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In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

Yersinopine, a nicotianamine-like metallophore, was recently identified through biochemical analyses, but its in vivo production and functional role remain uncharacterized. In Yersinia pseudotuberculosis and its recent descendant Yersinia pestis, the cnt operon (cntPQRLMI) putatively encodes the biosynthesis and transport of yersinopine. In Y. pestis, however, two frameshift mutations disrupt cntQ, which encodes the predicted permease for yersinopine-metal complexes. This pseudogenization raises critical questions about the functional relevance of yersinopine in these closely related species. Here, we show that cnt operon expression is repressed by the zinc uptake regulator Zur and that both Y. pestis and Y. pseudotuberculosis secrete yersinopine under zinc-limited conditions. Unexpectedly, the operon mediates iron uptake in Y. pseudotuberculosis but supports zinc acquisition in Y. pestis. Moreover, targeted disruption of cntQ in Y. pseudotuberculosis shifts metal specificity from iron to zinc, mimicking the Y. pestis phenotype. Collectively, our results suggest that a single pseudogenization event could rewire metal uptake specificity. Our findings illustrate how evolutionary genome reduction can reshape bacterial physiology.

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.

IntroductionHuman papillomaviruses (HPVs) are responsible for one-third of all cancers caused by infections. Most HPV studies focus on chronic infections and cancers, and we know little about the early stages of the infection. Our main objective is to better understand the course and natural history of cervical HPV infections in healthy, unvaccinated and vaccinated, young women, by characterising the dynamics of various infection-related populations (virus, epithelial cells, vaginal microbiota and immune effectors). Another objective is to analyse HPV diversity within hosts, and in the study population, in relation to co-factors (lifestyle characteristics, vaccination status, vaginal microbiota, human genetics).Methods and analysisThe PAPCLEAR study is a single center longitudinal study following 150 women, aged 18–25 years, for up to 2 years. Visits occur every 2 or 4 months (depending on HPV status) during which several variables are measured, such as behaviours (via questionnaires), vaginal pH, HPV presence and viral load (via qPCR), local concentrations of cytokines (via MesoScale Discovery technology) and immune cells (via flow cytometry). Additional analyses are outsourced, such as titration of circulating anti-HPV antibodies, vaginal microbiota sequencing (16S and ITS1 loci) and human genotyping. To increase the statistical power of the epidemiological arm of the study, an additional 150 women are screened cross-sectionally. Finally, to maximise the resolution of the time series, participants are asked to perform weekly self-samples at home. Statistical analyses will involve classical tools in epidemiology, genomics and virus kinetics, and will be performed or coordinated by the Centre National de la Recherche Scientifique (CNRS) in Montpellier.Ethics and disseminationThis study has been approved by the Comité de Protection des Personnes Sud Méditerranée I (reference number 2016-A00712-49); by the Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé (reference number 16.504); by the Commission Nationale Informatique et Libertés (reference number MMS/ABD/AR1612278, decision number DR-2016–488) and by the Agence Nationale de Sécurité du Médicament et des Produits de Santé (reference 20160072000007). Results will be published in preprint servers, peer-reviewed journals and disseminated through conferences.Trial registration number NCT02946346; Pre-results.