LA TRAPPE IONIQUE ET L'IONISATION ELECTROSPRAY (UN NOUVEAU POTENTIEL POUR LA CARACTERISATION DES BIOMOLECULES. CARACTERISATION DE BIOMOLECULES PRESENTES EN FAIBLE QUANTITE PAR SPECTROMETRIE DE MASSE ET SPECTROMETRIE DE MASSE MULTIPLE (MS N))

LA RECENTE INNOVATION INSTRUMENTALE DE LA SPECTROMETRIE DE MASSE A PERMIS D'ETENDRE LES POSSIBILITES DE LA TECHNIQUE D'ANALYSE EN AMELIORANT LES PERFORMANCES, ET EN ACCEDANT GRACE A LA SPECTROMETRIE DE MASSE TANDEM (MS 2) A DES INFORMATIONS STRUCTURALES FINES CARACTERISTIQUES DES MOLECULES ANALYSEES. L'OBJECTIF DE CE TRAVAIL A ETE D'EVALUER LES POSSIBILITES D'UNE NOUVELLE GENERATION DE SPECTROMETRE DE MASSE CAPABLE D'EFFECTUER DES ANALYSES MS N ; L'ESQUIRE-LC CONSTITUE D'UN ANALYSEUR TRAPPE IONIQUE (IT) COUPLE A UNE SOURCE ELECTROSPRAY (ES). LA PREMIERE PARTIE DU MANUSCRIT PRESENTE LA CARACTERISATION DES BIOMOLECULES PAR MS ET MS N ET PRECISE LES INFORMATIONS STRUCTURALES OBTENUES (CHAPITRE I). APRES AVOIR OPTIMISE L'ESQUIRE-LC (CHAPITRE II), NOUS AVONS ADAPTE DES STRATEGIES D'ANALYSES NANOES-MS N A CHAQUE ETUDE ABORDEE DANS LE CHAPITRE III. NOUS AVONS AINSI MIS EN EVIDENCE, DES MODIFICATIONS PROTEIQUES (ANALYSE PROTEOMIQUE ET ANALYSE DE NOUVEAUX VARIANTS D'HEMOGLOBINE), CARACTERISE UNE MODIFICATION ENGENDREE PAR UN INHIBITEUR SUR UNE ENZYME (L'ADOHCY HYDROLASE), SEQUENCE DE NOVO UN PEPTIDE N-BLOQUE ISSU DE LA DROSOPHILE ET ACCEDE AUX STRUCTURES COMPLEXES DES GLYCOSYLATIONS PROTEIQUES. LA DEUXIEME PARTIE DU MANUSCRIT PRESENTE DES ETUDES DE CARACTERISATION D'EDIFICES NON-COVALENTS BIOLOGIQUES QUI NECESSITENT DES PRECAUTIONS D'ANALYSE MS MINUTIEUSES (CHAPITRE I). NOTRE OBJECTIF ETAIT DE DETERMINER PREALABLEMENT LES CONDITIONS INSTRUMENTALES ADEQUATES DE L'ESQUIRE-LC, PROPICES AU MAINTIEN DES INTERACTIONS FAIBLES EN PHASE GAZEUSE ET A L'INTEGRITE DE L'EDIFICE NON-COVALENT (CHAPITRE II). TRES PRISEE PAR LES BIOLOGISTES, LA MS SUPRAMOLECULAIRE NOUS A PERMIS D'EVALUER LA SELECTIVITE DU CIS PLATINE POUR L'ADN (CHAPITRE III). CE TRAVAIL SOULIGNE L'INTERET DE LA MS N PAR ES-IT POUR L'ELUCIDATION STRUCTURALE DES BIOMOLECULES. IL EST CEPENDANT PRIMORDIAL DE MAITRISER LES DIFFERENTS PARAMETRES INSTRUMENTAUX QUI FACILITENT L'OBTENTION DES INFORMATIONS MAIS SURTOUT LEUR QUALITE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

International audienceMore than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

International audienceMore than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15–RBM39 interaction, we solved the DCAF15–DDB1–DDA1–indisulam–RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets

PAX8 and MECOM are interaction partners driving Ovarian Carcinogenesis

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian carcinogenesis. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. This includes PRDM3 (also called MDS1-EVI1) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulate a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, PTXs and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in Ovarian Cancers and we show that the MECOM locus and PAX8 sustain in-vivo ovarian cancer growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer