Evidence for the Formation of a Covalent Thiosulfinate Intermediate with Peroxiredoxin in the Catalytic Mechanism of Sulfiredoxin

International audienceThe typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sul-fenic acid. Sulfiredoxin was originally proposed to operate by cova-lent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-re-ducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of 2 min 1 , for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoi-chiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thio-sulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway

Projet PRODIADOM "Promouvoir la dialyse à domicile"

(The main text is available only in French) PRODIADOM is an innovative solution designed to help professionals to develop home dialysis. By the moment only available for Peritoneal Dialysis (very soon as well for Home Haemodialysis), PRODIADOM is a very simple, practical, useful and user-friendly website. Nephrologists will find in guidelines, formations, practice-sheets, decisions-trees, decision-making process in complex situations, functional evaluations and tests in Peritoneal Dialysis, their indications and practical executions. PRODIADOM’s ambition is to become a practical reference for doctors and nurses who want to start a home dialysis program in their renal unit. Created by nephrology experts, the website access is free.PRODIADOM est une solution innovante destinée à aider les professionnels qui souhaitent développer la dialyse à domicile. Dans un premier temps disponible pour la dialyse péritonéale (bientôt disponible pour l’hémodialyse à domicile), PRODIADOM propose un site Web qui se veut simple, pratique, utile et convivial. Les praticiens pourront y trouver des formations, des guides, des fiches pratiques, des arborescences décisionnelles, des conduites à tenir face à des situations inhabituelles, des recommandations, tous les protocoles utiles pour la dialyse péritonéale, toutes les explorations fonctionnelles péritonéales courantes, avec leurs indications et leur réalisation pratique. PRODIADOM a l’ambition de devenir la référence des professionnels médicaux et paramédicaux qui souhaitent démarrer un programme de dialyse à domicile. Conçu par des experts, son accès est gratuit

High Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody

International audienceMonoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes (CQAs) that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the Complementarity-Determining Regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb collected peptide and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs

Low incidence of SARS-CoV-2, risk factors of mortality and the course of illness in the French national cohort of dialysis patients

International audienceThe aim of this study was to estimate the incidence of COVID-19 disease in the French national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. Our study included all patients on dialysis recorded in the French REIN Registry in April 2020. Clinical characteristics at last follow-up and the evolution of COVID-19 illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for SARS-CoV-2. A total of 1,621 infected patients were reported on the REIN registry from March 16th, 2020 to May 4th, 2020. Of these, 344 died. The prevalence of COVID-19 patients varied from less than 1% to 10% between regions. The probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. Dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. Mortality in diagnosed cases (21%) was associated with the same causes as in the general population. Higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. Being treated at a selfcare unit was associated with a lower risk. Thus, our study showed a relatively low frequency of COVID-19 among dialysis patients contrary to what might have been assumed