Candice Sartre's thesis defense (LBPA)

Jury

• Rapporteur and Examiner : Gervaise Loirand, Directrice de recherche, Université de Nantes
• Rapporteur and Examiner : Michel Franco, Directeur de Recherche, Université côte d’azur
• Examiner : Herman Van Tilbeurgh, Professeur des universités, Université Paris Saclay
• Examiner : Frank Lezoualc’h, Directeur de recherche, Université de Toulouse

Supervisor

• Director of thesis : Jacqueline Cherfils, Directrice de la Recherche, CNRS
• Supervisor : Yann Ferrandez, Chargé de recherche, ENS-Paris-Saclay

Abstract

Titel : Regulation and inhibition of the exchange factor Epac1: a therapeutic target in heart failure

EPAC proteins are guanine nucleotide exchange factors (GEFs), directly activated by the cyclic AMP second messenger (cAMP), which activate small Rap GTPases at the periphery of cell membranes.
The EPAC1 protein, mainly expressed in the heart, plays an important role in cardiovascular physiology and pathophysiology, and thus constitutes an attractive therapeutic target. Activation of EPAC1 by cAMP results in a large-amplitude conformation change. Recent work by the team has revealed that EPAC1 is also regulated by biological membranes. More than just a meeting place between the GEF and its GTPase, membranes play an essential role in the activation of small GTPases.
This thesis project has two objectives: understand how this membrane environment contributes, in cooperation with cAMP, to the activation of the EPAC1 protein. To do this, I quantified the biochemical modalities of activation of EPAC1 by the cAMP and membrane, and undertook to study its different structural states by HDX-MS. Understand the mechanism of action of an allosteric EPAC1 inhibitor, CE3F4, taking into account the contribution of the membrane.
My results confirm that membranes induce a significant increase in the affinity of EPAC1 for cAMP from 30 µM to 70 nM. They also show that EPAC1 is activated by membranes even in the absence of cAMP, that cAMP-bound EPAC1 associated with the membrane is much more active than in the presence of cAMP alone, and that EPAC1 exists in at least 4 different conformations whose activity depends on the cAMP and membrane. Then, I showed that only intermediates activated by cAMP (cAMP and cAMP+membrane) are targeted by CE3F4.
These results suggest that in cells, EPAC1 must be pre-activated by the membrane to  respond to cAMP, and that only fully activated EPAC1 can be inhibited by CE3F4. These results will be important for the development and improvement of new therapeutic molecules.

Crédits illustration : Jérôme Fouber