Conception rationnelle de peptides inhibiteurs et le développement de myopathies viscérales
INSTITUT
Nom : PhyMedExp-INSERM U1046
Adresse : CHU Arnaud de Villeneuve -Avenue doyen Giraud-Montpellier
Téléphone : 0467415240
Email : u1046@inserm.fr
TUTEUR
Nom : MARCHAL Stéphane
Fonction : CRCN
Téléphone : 0467415224
Email : stephane.marchal@inserm.fr
STAGE
Sujet :
Conception rationnelle de peptides inhibiteurs et le développement de myopathies viscérales
Description courte :
Digestive motility disorders are frequently encountered in pediatric and aging population. Studying the development and the physiological dedifferentiation (plasticity) of the gastrointestinal smooth muscle, our team demonstrated that the smooth muscle cell (SMC) plasticity process was unbalanced in Chronic Intestinal PseudoObstruction (CIPO) patients. We identified the function of the RNA-binding protein RBPMS2 in the plasticity of SMCs and a positive correlation between high level of RBPMS2 in CIPOs.
The objectives of the Master proposal aim to develop different Proteolysis-Targeting Chimeras (PROTACs) peptides from the RBPMS2 stapled peptides that have been identified by the consortium (PHYMEDEXP and IBMM) and to test the effectiveness of these molecules on the degradation of RBPMS2. This work paves to the development of specific inhibitory approach in the context of smooth muscle alterations and promising treatments for pediatric and adult CIPO patients.
Activité :
The project carried out during the M2 internship aims to develop different PROTAC peptides from the RBPMS2 stapled peptides that have been identified by the consortium (IBMM and PHYMEDEXP) and to test the effectiveness of these molecules on the degradation of RBPMS2.
1/ Synthesis of PROTAC-RBPMS2:
The precursor linear peptide sequences will be synthesized according to conventional microwave assisted solid phase peptide synthesis (SPPS). After development of the linear peptide, the staple will be generated on the solid support. All peptide sequences will be capped at the C-terminus by an acetyl group. After identification of stapled RBPMS2 peptides that are able to efficiently bind to the protein, they will be linked via a spacer to an E3 ligase ligand (CRBN and VHL) to develop PROTAC-RBPMS2 compounds.
2/ Evaluation of the impact of PROTAC-RBPMS2 compounds using model cells expressing RBPMS2.
PROTAC-RBPMS2 compounds will be used on different cell lines expressing exogenous and endogenous RBPMS2 in order to evaluate the capacity of PROTAC-RBPMS2 compounds to degradate RBPMS2 protein in concentration- and time-dependent manner by western blot analysis. To ensure the proteasome-dependence of RBPMS2 degradation, cells will be pre-treated with specific proteasome inhibitor such as Epoximicin. To evaluate that PROTAC-RBPMS2 compounds achieve intracellular access without direct membrane disruption, we will perform lactate dehydrogenase (LDH) release assays using the LDH Cytotoxicity Detection Kit. The cytotoxicity of PROTACs-RBPMS2 treated cells will be assessed by colorimetric method (crystal violet or Annexin V assay). Most efficient PROTAC-RBPMS2 compounds in a dose-response concentration will be tested on RBPMS2-expressing SMC models or CIPO SMC cultures. We will evaluate the differentiation status using specific SMC markers (αSMA, SM22, CALPONIN, CALDESMON, MYOCARDIN) and the proliferative profile (Ki67 rate, MTT cell proliferation assays) of these treated SMCs. Forthcoming results should have implications for the potential study and promising treatments for pediatric and adult CIPO patients.
Compétences requises : Cell culture, biochemistry (western blot, immunoprecipitation), imaging
Encadrement : Group staff, team meeting
Gratification de stage : Sur fonds propres du laboratoire
Tutelle de stage : Bourse de Master KIM “Biomarkers & Therapy” 2021-2022 – Université de Montpellier
