Etude structurale et fonctionnelle de la protéine arginine méthyltransférase CARM1

La protéine CARM1 "Coactivator-Associated aRginine Methyltransferase 1" a été initialement identifiée pour sa fonction coactivatrice de la transcription impliquant différents récepteurs nucléaires des hormones. Cette voie d activation de la transcription est un processus régulé par plusieurs étapes et différentes cascades de signalisation impliquant de nombreux corégulateurs. Dans ce contexte, deux cibles privilégiées ont été identifiées pour l'activité de méthylation de CARM1: l'histone H3 et la protéine CBP. CARM1 est également impliquée dans d autres processus, comme la maturation des ARN et la transmission du signal cellulaire. Une dérégulation de ces différents processus pouvant induire certains cancers, CARM1 constitue une des nouvelles cibles potentielles en chimiothérapie. Les résultats obtenus durant cette thèse sont répartis en quatre chapitres. Le chapitre 3 présente les structures cristallographiques de domaines isolés de CARM1 résolues seules ou en complexe avec différents cofacteurs. Le chapitre 4 porte sur les études fonctionnelles en solution par la comparaison de différentes constructions et de mutants ponctuels. Ces études mettent en évidences des zones et des résidus cruciaux à l activité de CARM1. Le chapitre 5 détaille l étude des interactions entre CARM1 et différents substrats. Ceci a été fait par différentes approches : l étude de protéines complètes (histone H3 et CBP), de fragments de ces protéines et une approche qui consiste à fixer de façon covalente des peptides de l histone H3 à un complexe binaire CARM1-cofacteur. Le chapitre 6 porte sur la recherche d inhibiteurs spécifiques de CARM1, travaux effectués dans le cadre de collaborations.The protein CARM1 "Coactivator-Associated aRginine Methyltransferase 1" was initially identified for its activity to coactivate transcription under the regulation of some nuclear receptors. This kind of activation process is regulated by a large number of coactivators involved at different stages. In this context, CARM1 was identified to methylates two major targets: histone H3 and CBP. CARM1 is also involved in other biological events like RNA processing and signal transmission. Disturbances of these different processes can induce cancers and CARM1 is by the way a new potential pharmacological target in chemotherapy. The results obtained during this thesis work are divided in four chapters. Chapter 3 presents the crystallographic structures of isolated domains of CARM1, alone or in complex with different cofactors. Chapter 4 describes the functional studies in solution by comparisons of different constructions and mutant forms. These studies highlight the crucial role of different areas and residues for the CARM1 activity. Chapter 5 details studies of interactions between CARM1 and selected target substrates (histone H3 and CBP). It has been done by three different approaches: interactions with full length proteins, fragments of these proteins and an approach consisting of covalently bind H3 peptides to a binary complex CARM1-cofactor. Chapter 6 presents a collaborative work done to discover CARM1 specific inhibitors.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering

Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1

Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes