Membrane contact sites (MCSs) enable specific lipid exchange between organelles. Our recent results on the archetypical OSBP/VAP complex suggest that the architecture and dynamics of MCS are influenced by intrinsically disordered regions (IDRs). These overlooked structural attributes enable formation of MCS of adjustable thickness and reduce protein crowding. These effects are likely to be general given the abundance of IDRs in MCS proteins. We posit that IDRs guarantee the lateral and/or vertical flexibility of proteins. We will dissect the link between these characteristics and the function, dynamics and organization of MCSs, by using a multidisciplinary and multi-scale approach involving quantitative and super-resolution cell imaging, in vitro reconstitution of membrane systems, structural analysis by cryo-electron tomography, and the development of innovative pharmacological tools. This project will offer new perspectives on the efficiency, plasticity and specificity of MCSs.

This project aims at investigating the chemistry of a-aminoendoperoxides. This class of reactive molecules has a great potential for the development of powerful and original methods in organic synthesis, and for applications to the synthesis of complex nitrogen-containing polycyclic systems. Their reactivity has been scarcely studied so far and remains essentially unexplored. The foundations of our proposition lie on the scientific expertise of the three partners, whose know-how will be engaged in a synergic fashion. Beyond the synthesis of aminocyclopropanes by the Kulinkovich-de Meijere reaction, that we know well, and the electrochemical oxidation method that we have recently developed, we will focus a great deal of our efforts to new, particularly ambitious aspects of this chemistry: the improvement and the extension of the Kulinkovich-de Meijere reaction using the advantages of electrochemistry, the generalisation of the controlled oxidation of cyclopropylamines to other types of cyclopropanes with a low ionisation potential, or the development of alternative methods relying on photocatalytic processes, even more liable to fulfil the requirements of the current global context targeted towards sustainable development. We will synthesise a range of novel natural product-like or drug-like molecules. Their biological activities will be systematically scrutinised.

Many pathogens manipulate the cAMP intracellular signaling of host cells to promote their survival and proliferation in hosts. Bacterial ExoY-like virulence factors represent a new atypical subfamily of nucleotidyl cyclase (NC) toxins. Exoenzyme Y (ExoY) was first identified as a toxin secreted via a type 3 secretion system (T3SS) by Pseudomonas aeruginosa, a major opportunistic and nosocomial human pathogen. P. aeruginosa is also responsible for progressive and severe chronic lung infections in patients with cystic fibrosis. ExoY is present in 90-98% of clinical isolates of P. aeruginosa, which suggests an important role in bacterial pathogenicity, but its role in P. aeruginosa infections remains poorly understood. Poorly characterized ExoY-related enzymatic modules or effector proteins have also been found recently in several toxins produced by various Gram-negative bacteria representing emerging human or animal pathogens. Some of these enzymes were shown to be essential for bacterial virulence. We have recently shown that these NC toxins are potently activated within the host target cells by using an original eukaryotic cofactor that is actin. Yet, our new preliminary data suggest that they may differ significantly in their substrate selectivity, their interaction with actin and activation mechanisms, impact on the actin cytoskeleton dynamics, and subcellular localizations. As a consequence, they likely perturb different biological processes in infected host cells. We aim here to characterize at the molecular and structural level and in cellular infection models the functional specificities, precise role and virulence mechanisms of this novel class of actin-activated NC toxins in bacterial infections by P. aeruginosa and various pathogenic Gram-negative organisms. To decipher their structure-function relationships we will analyze several representative members of this subfamily of actin-activated NC toxins. Our main goals are to characterize in vitro and in cells their functional specificities and impact on actin self-assembly dynamics. We will study the structural bases for their activation mechanisms and enzymatic specificities, and search for inhibitors. We will characterize ExoY phenotype and prevalence in the strains included in the P. aeruginosa reference panel collection. We will study ExoY effects on phagocytosis, inflammation, mucus production, or the integrity of cell-cell junctions in P. aeruginosa-infected cells to determine the importance of ExoY in the modulation of host airways innate immunity. We will finally analyze possible interplays between ExoY/ExoY-like virulence factors and other bacterial virulence factors that affect the actin cytoskeleton. Collectively, our proposed research will be innovative in several aspects: it will expand the fundamental knowledge on Pseudomonas aeruginosa T3SS effector virulence mechanisms in P. aeruginosa infections; it will help to identify the functional specificities of ExoY-related toxins recently found in emergent human or animal proteobacterial pathogens, and will eventually explore their potential as drug target. Finally, our project may help to understand better the pathogenicity of P. aeruginosa in diseases such as cystic fibrosis for which there is yet no effective therapy.

The arsenal of advanced spectroscopic techniques in the gas phase is able to reveal the intrinsic properties of a molecular system (structure, dynamics...) at a unique level of detail, provided that it is prepared in cold, isolated conditions. The VAPOBIO project aims at developing a new vaporization technique based on a supercritical fluid (CO2) capable to produce such cold isolated neutral species for spectroscopic analysis, and then applying it to non-volatile biomolecular systems. Two vaporization sources, one continuous and one pulsed, will be built to study these systems by ionization-based spectroscopies and supercritical fluid chromatography. Applications in basic research are targeted along three scientific axes: A. The analysis of complex mixtures like extracts of natural products; B. The detailed structural characterization of biopolymers (peptides, sugars) isolated, or in supramolecular assemblies; C. Photophysical processes such as photofragmentation and ultrafast dynamics of selected biomolecular systems. The vaporization sources developed in this project will enable the combination of supercritical fluid chromatography with gas phase advanced spectroscopies, thus creating a powerful analytical tool potentially able to trigger several breakthroughs beyond the scientific fields directly targeted by this project.