CD5, an Undercover Regulator of TCR Signaling

T cells are critical components of adaptive immunity. As such, their activation is regulated by the T cell receptor (TCR) that constantly scan peptides associated with major histocompatibility complexes (MHC). TCR engagement initiates a series of molecular events leading to cytokine secretion, proliferation, and differentiation of T cells. As a second coincident event, activation of co-stimulatory molecules, such as CD28, synergize with the TCR in order to prolong and/or amplify intracellular signals. With the recent advances in immunotherapies targeting T cells, co-inhibitory receptors are of growing interest for immunologists due to their potential modulatory properties on T cell functions. However, special attention should be dedicated to avoid unwanted clinical outcomes (1). In particular, Manichean categorization of receptors based on incomplete functional knowledge can lead to an over-simplistic view of complex cellular regulations. Thus, analysis of the functions that characterize these receptors in diverse physiological contexts remains essential for their rational use in therapeutic protocols. Here we focus on CD5, a transmembrane receptor that regulates T cell functions and development but remains poorly characterized at the molecular level. We will review its roles in physiological conditions and suggest potential molecular effectors that could account for CD5-dependent regulation of TCR signaling

Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

International audienceIn Brief 20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled pro-teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFN␥ or amplified in a 20% O 2 environment. Graphical Abstract Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O 2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity

ETUDE STRUCTURALE DE LA PROTEINE FUR (FERRIC UPTAKE REGULATION) D'ESCHERICHIA COLI PAR SPECTROMETRIE DE MASSE

LA PROTEINE FUR (FERRIC UPTAKE REGULATION) REGULE LES MECANISMES D'ACQUISITION DU FER CHEZ LES BACTERIES GRAM NEGATIVES COMME ESCHERICHIA COLI. LE FER EST NECESSAIRE A LA CROISSANCE DE PRESQUE TOUS LES ORGANISMES VIVANTS, ET LES BACTERIES ONT DEVELOPPE DES SYSTEMES COMPLEXES POUR L'ACQUERIR. NEANMOINS, A FORTE CONCENTRATION, IL CATALYSE LA FORMATION DE RADICAUX LIBRES, ET DEVIENT TOXIQUE. LA CONCENTRATION INTRACELLULAIRE EN FER DOIT DONC ETRE STRICTEMENT CONTROLEE. LORSQUE CELLE-CI DEVIENT TROP ELEVEE, FUR FIXE DES IONS FE(II) ET EST AINSI ACTIVEE : ELLE SE FIXE ALORS SUR LA REGION PROMOTRICE DES GENES IMPLIQUES DANS L'ACQUISITION DU FER. LA PROTEINE INHIBE AINSI L'EXPRESSION DE CES GENES, ET LIMITE L'ENTREE DE FER DANS LA BACTERIE. FUR EST UNE PROTEINE HOMODIMERIQUE DE 2 FOIS 17 KDA. ELLE POSSEDE UN SITE DE FIXATION DU FE(II) PAR MONOMERE, ET CONTIENT AUSSI DU ZN(II). SA STRUCTURE TRIDIMENSIONNELLE N'EST PAS CONNUE, ET DANS CETTE ETUDE, LA SPECTROMETRIE DE MASSE A ETE UTILISEE COMME OUTIL D'ANALYSE POUR OBTENIR DIVERSES INFORMATIONS STRUCTURALES CONCERNANT CETTE PROTEINE. DES MESURES EN CONDITIONS NON DENATURANTES ONT MONTRE QUE LA PROTEINE CONTIENT UN SITE A ZINC PAR MONOMERE. PAR AILLEURS, UNE APPROCHE COMBINANT MODIFICATION CHIMIQUE DES CYSTEINES ET ANALYSE PAR SPECTROMETRIE DE MASSE A PERMIS D'ETABLIR QUE LES CYSTEINES 92 ET 95 SONT LIGANDS DU ZINC. DES METHODES DE RETICULATION CHIMIQUE PROTEINE-PROTEINE ONT ETE EMPLOYEES POUR LOCALISER LES INTERACTIONS MONOMERE-MONOMERE AU SEIN DU DIMERE DE FUR. ENFIN, LES INTERACTIONS PROTEINE-ADN ONT EGALEMENT ETE ETUDIEES. LA MODIFICATION CHIMIQUE DIFFERENTIELLE DES LYSINES, EN PRESENCE OU NON D'ADN, A MONTRE QUE LA LYSINE 76 EST PROTEGEE APRES LIAISON DE LA PROTEINE A L'ADN, SUGGERANT QUE CE RESIDU APPARTIENT A LA ZONE D'INTERACTION. LA METHODE DES ECHANGES HYDROGENE-DEUTERIUM, ASSOCIEE A LA SPECTROMETRIE DE MASSE, A PERMIS DE CARACTERISER LES CHANGEMENTS CONFORMATIONNELS DE LA PROTEINE LORS DE LA FIXATION DU METAL OU DE L'ADN. DES EXPERIENCES DE PONTAGE PROTEINE-ADN PAR LASER UV, DESTINEES A LOCALISER LES POINTS DE CONTACT ENTRE LES DEUX ESPECES, SONT EGALEMENT DECRITES. SUR LA BASE DES RESULTATS OBTENUS ET DES PREDICTIONS DE STRUCTURE SECONDAIRE DE FUR, UN MODELE D'INTERACTION PROTEINE-ADN FAISANT INTERVENIR UN MOTIF HELICE-COUDE-HELICE ATYPIQUE EST PROPOSE.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Développement de méthodes quantitatives sans marquage pour l'étude protéomique des cellules endothéliales

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

An Optimized Strategy for ICAT Quantification of Membrane Proteins*

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General Repression of RNA Polymerase III Transcription Is Triggered by Protein Phosphatase Type 2A-Mediated Dephosphorylation of Maf1

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Urine in clinical proteomics.

International audienceUrine has become one of the most attractive biofluids in clinical proteomics as it can be obtained non-invasively in large quantities and is stable compared with other biofluids. The urinary proteome has been studied by almost any proteomics technology, but mass spectrometry-based urinary protein and peptide profiling has emerged as most suitable for clinical application. After a period of descriptive urinary proteomics the field is moving out of the discovery phase into an era of validation of urinary biomarkers in larger prospective studies. Although mainly due to the site of production of urine, the majority of these studies apply to the kidney and the urinary tract, but recent data show that analysis of the urinary proteome can also be highly informative on non-urogenital diseases and used in their classification. Despite this progress in urinary biomarker discovery, the contribution of urinary proteomics to the understanding of the pathophysiology of disease upon analysis of the urinary proteome is still modest mainly because of problems associated to sequence identification of the biomarkers. Until now, research has focused on the highly abundant urinary proteins and peptides, but analysis of the less abundant and naturally existing urinary proteins and peptides still remains a challenge. In conclusion, urine has evolved as one of the most attractive body fluids in clinical proteomics with potentially a rapid application in the clinic

Opposing regulatory functions of the TIM3 (HAVCR2) signalosome in primary effector T cells as revealed by quantitative interactomics

International audienceDeciphering how T-cell antigen receptor signals are modulated by coinhibitors is a fundamental goal in immunology and of considerable clinical interest because blocking coinhibitory signals via therapeutic antibodies have become a standard cancer immunotherapeutic strategy. Most of the attention devoted to T-cell immunoglobulin and mucin domain-3 (TIM3; also known as HAVCR2 or CD366) molecules stems from their expression on exhausted T cells in settings of chronic viral infection and tumors. Moreover, T cells expressing high levels of both PD-1 and TIM3 coinhibitors appear more dysfunctional than those expressing PD-1 alone. Combination therapies intending to block both PD-1 and TIM3 are thus actively being explored in the cancer treatment setting. Upon interaction with Galectin-9 (GAL-9) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1), the tyrosines found in the TIM3 cytoplasmic tail are phosphorylated.1 Because these conserved tyrosines do not form a recognizable inhibitory signaling motif, the mechanism by which TIM3 transmits inhibitory signals has not been elucidated. Paradoxically, TIM3 also has costimulatory activity in T cells.2,3 Published biochemical studies attempting to unveil the mode of action of TIM3 have relied on approaches addressing one candidate effector at a time with limited quantitative insight, and most used transformed cells. Using mice expressing an affinity Twin-Strep-tag (OST) at the TIM3-protein C-terminus (TIM3OST mice) (Figs. 1a and S1a) and affinity purification coupled with mass spectrometry (AP-MS), we herein defined the composition and dynamics of the signaling protein complex (signalosome) used by TIM3 in primary effector T cells. These results provide a more complete model of TIM3 signaling and explain its paradoxical coinhibitory and costimulatory function

Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33

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