Résumé:

Platelets are small blood cells that help stop bleeding by forming clots, but they can also cause life-threatening, clots that stop blood flow to tissues and organs leading to heart attacks or strokes. Most current anti-platelet drugs reduce clotting, but also increase the risk of bleeding, which is a major problem.

In this project, researchers present a new strategy to prevent harmful clot formation without severely affecting clotting. They developed innovative bispecific antibody molecules called cis-acting platelet receptor inhibitors (CAPRIs) that work by limiting specific platelet activation signals rather than completely blocking these critical pathways. CAPRIs bring together a natural inhibitory receptor on platelets (G6B) with activating receptors that are responsible for thrombus formation, including the collagen and immunoglobulin receptors GPVI and FcgRIIA (CD32A), respectively.

By doing this, CAPRIs effectively prevented platelet activation caused by collagen or immune complexes in several disease-related models, including arterial thrombosis, heparin- and vaccine-induced thrombocytopenia (reduced platelet count) and thrombosis (HIT and VITT), antiphospholipid syndrome (APS), and sepsis. Importantly, these effects were seen under conditions that closely mimic blood flow in arteries.

Overall, this research reveals that CAPRIs can selectively block harmful platelet activation pathways. This approach could lead to a new class of anti-thrombotic treatments that are effective at preventing pathological blood clotting while potentially causing fewer bleeding side-effects than current therapeutics.

Projet inter-institut IM2B - ICI

Lanthanides (Ln) also known as Rare earth elements (REE) possess chemical, magnetic, luminescent and electric properties of high interest in the industry, medicine and renewable energy. REE are critical resources for the high technologies of ever-growing interest (Figure1). REE are common metals (0.5 to 60 ppm) of the Earth’s crust but are most often found in mixture. Due to their close physicochemical properties, their selective extraction is a challenge. Classic extraction and separation methods required several steps (up to 100) and generate a lot of pollutant wastes. Alternative methods to obtain more efficient and more sustainable REE extraction that selective are highly needed. Bio-inspired tools are the actual most promising options. Indeed, in the past decades, studies on bacterial system suggest that REE biochemistry could solve the selectivity issue. Only the light REE (La to Eu) are naturally used by bacteria, indicating that bacteria are able to selectively discriminate between REE. More broadly, those discoveries open a new field of research for which much is to be uncovered.

The first REE-interacting protein identified was the methanol dehydrogenase (MDH) of a methylotroph bacteria. Further investigations revealed that methylotroph bacteria also possess other REE interacting proteins, consequently most of the work focused on methylotroph’s proteins. To date three binding sites of REE-interacting protein have been characterized (XoxF, LanM and LanD), some present a preference trend toward light REE but limit of the selectivity is not fully understood yet. Conservation analysis revealed that MDH like proteins are more widespread among bacteria than initially thought. This opened the way for new investigations in other bacteria. Interestingly, for at least some organisms carrying the two forms of methanol/alcohol desydrogenases, i.e. calcium (Ca²⁺) or REE dependent. The regulation allowing the bacterial adaptation leading to the expression switch in between the two forms of MDH/ADH, is under the control of a complex regulatory network involving two-component systems (TCS), and in some case other proteins. Importantly, TCS involved in the REE adaptation seems to be among the most selective and vary from organisms revealing that selective detection mechanisms might differ between bacteria. None of those detailed molecular mechanism involved in the REE detection has been uncover.

Few recent studies done by IPM collaborator P. Billard (Univ. Lorraine) in the non-methylotroph bacteria Pseudomonas putida, confirmed the broader conservation of REE proteins. Among them the two-component system (TCS) called PedS2/PedR2, in charge of the regulation switch between the two alcohol deshydrogenases (REE or Ca²⁺ dependent), for which the detailed REE detection mechanism is not fully understood (Figure2). The regulon of this TCS is not identified either. Deciphering the molecular mechanism used by this TCS to first sense the REE, either indirectly or directly, discriminate and then regulate the targeted genes is key to broaden the knowledge on REE adaptation. 

The aim of this project is to characterize for the first time the molecular mechanism involved in the bacterial REE sensing using P. putida as model. To this end, the strategy is to purify PedS2 protein and identify the REE detection mechanism using a combination of approaches to provide insights on the potential key residues involved in the REE sensing. Following the PedS2 characterization, the goal is to further investigate the PedR2 regulon and its binding properties. Thanks to the IM2B grant, we have been able to test in vivo potential PedS2 interacting partner, validate the PedS2 topology (Figure3), notably the sensor loop, purified the PedS2 sensor loop, initiate the in vitro characterization of PedS2 REE detection mechanism with preliminary results suggesting a direct binding. In other proteins, REE binding is coordinated by carboxylate residues. Next, we realized an in silico analysis to predict potential residues involved in the binding (Figure3). 

The bio-conversion of CO₂ by the Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) reaches 1014 kilograms per year. The carboxylation of Ribulose 1,5 bisphosphate (RuBP) activity of RuBisCO competes with its oxygenation activity. However, the diffusion of CO₂ in water is much less efficient than that of O₂, and the majority of inorganic carbon is in the form of bicarbonate in the aquatic environments. The success of photosynthetic microorganisms relies in their CO₂ concentration mechanisms (CCM) that induces a flux of inorganic carbon near RuBisCO. Contrary to higher plant, eukaryotic microalgae such as the model green algae Chlamydomonas reinhardtii possess biophysical CCMs which involves a peculiar supramolecular organisation and specific location of RuBisCO as well as bicarbonate transporters and carbonic anhydrases. RuBisCO is found in two distinct locations in C. reinhardtii: either diffused in the chloroplast stroma or condensed in a liquid-liquid separated phase (LLSP) named Pyrenoid. The proportion of diffused vs condensed RuBisCO varies from 60%/40% under condition of high partial pressure of CO₂ (P_CO2) to 20%/80% under low P_CO2. While numerous studies have investigated the carboxylation vs oxygenation activities of isolated RuBisCO or in vivo, the effect of condensation on the enzyme activity has been poorly investigated. The objective of this project is to correlate RuBisCO condensation to its activity. This is declined in two main strategies: 1- in-vitro reconstituted LLPS with recombinant RuBisCO and its scaffold protein EPYC1 and a structure-function investigation, and 2- in-vivo quantification of carboxylation and oxygenation activities under conditions in which RuBisCO is mainly diffused or mainly condensed.

A new tool coming from nanobodies^R has emerged: Megabodies^R.

Megabodies ^R are constructed by grafting a nanobody to a scaffold protein, enhancing the molecular weight while retaining the affinity and specificity of binding towards the target.

The purpose of those megabodies is to overcome two major obstacles that limits cryo-EM resolution : size particles and preferential orientation at the water-air interface (Uchański et al., 2021).

This project aims at developing a methodology to design megabodies ^R from existing nanobodies^R.

The severe acute respiratory syndrome coronavirus (SARS-CoV/CoV-2) genome encodes 16 non-structural proteins (nsps), which coordinate cell remodeling, virus replication and participate in viral evasion. Notably, nsp3 contains a protein module termed Macro domain, which carries IFN antagonist activity that interferes with host innate immunity response. This domain is able to bind and hydrolyze ADP-ribose derivatives. This activity is correlated to viral escape and thus makes Macro domains a valuable therapeutic target. In the present paper, we report a SARS-CoV Macro domain structure in complex with a MOPS molecule. Based on our structural data, molecular docking was performed on a set of MOPS analogs in the ADP-ribose binding pocket. We present an ELISA-based assay to select hits based on the inhibition of recombinant SARS-CoV/CoV-2 Macro domain-ADP-ribose complex formation. Among the tested analogs, MOPSO and CAPSO are the more efficient in inhibiting ADP-ribose-binding. Structural analysis of these molecules in the ADP-ribose pocket reveals potential interactions with amino acid residues involved in the coordination of ADP-ribose. Overall, these findings suggest that MOPSO and CAPSO bear potential to be used as a scaffold for the design of Macro domain-specific inhibitors.