Catherine Michaux

Biomineralization is a ubiquitous phenomenon that still fascinates the scientific community. Mineralized structures are most notably encountered in marine lifeforms, the shell or exoskeleton of which are formed by precipitating specific calcium carbonate (CaCO3) polymorphs, known as amorphous (ACC), vaterite, aragonite, and calcite. To control crystalline polymorphism in their shell layers, bivalves have evolved strategies involving ion-binding secretomes composed of shell matrix proteins (SMPs). Secreted in the prismatic layer of the pearl oyster Pinctada fucata, Aspein is an unusually acid-rich protein exclusively aspartic and famously recognized as the most acidic SMP known to date. Through in vitro crystallization experiments, Aspein was shown to select calcite over aragonite in modern seawater conditions. However, how it exerts its selectivity with respect to its structural properties remains enigmatic. By combining sequence-based predictions with all-atom molecular dynamics simulations, we unveiled that Aspein is an intrinsically disordered organic matrix protein (IDOMP) undergoing ion-specific chain collapse and phase separation. Thanks to its unique Asp density and conformational fuzziness, Aspein first sequesters Ca2+ then attracts CO32– ions before stabilizing ACC and calcite-like phases, while precluding the incorporation of Mg2+ to avoid aragonite formation. Indeed, via Asp-Ca2+ bridges, the coalescence of structure-less clusters into supersaturated protein-CaCO3 assemblies promotes calcitic ordering. Therefore, Aspein plasticity, condensation susceptibility, and aspartic density critically modulate the pathway that defines CaCO3 polymorphism by inhibiting nucleation and controlling the growth of biogenic minerals. From a broader perspective, our study underpins some of the molecular and physicochemical principles governing biomineralization processes mediated by IDOMPs, the overlooked yet prevalent role of which was mechanistically unexplored.

Biomineralisation is a ubiquitous phenomenon that still fascinates the scientific community. Mineralised structures are most notably encountered in marine lifeforms the shell or exoskeleton of which are formed by precipitating specific calcium carbonate (CaCO ₃ ) polymorphs, known as amorphous (ACC), vaterite, aragonite, and calcite. To control crystalline polymorphism in their shell layers, bivalves have evolved strategies involving ion-binding secretomes composed of shell matrix proteins (SMPs). Secreted in the prismatic layer of the pearl oyster Pinctada fucata , Aspein is an unusually acid-rich protein exclusively aspartic and famously recognised as the most acidic SMP known to date. Through in vitro crystallisation experiments, Aspein was shown to select calcite over aragonite in modern seawater conditions. However, how it exerts its selectivity with respect to its structural properties remains enigmatic. By combining sequence-based predictions with all-atom molecular dynamics simulations, we unveiled that Aspein is an intrinsically disordered organic matrix protein (IDOMP) undergoing ion-specific chain collapse and phase separation. Thanks to its unique Asp density and conformational fuzziness, Aspein first sequesters Ca ²⁺ then attracts CO ₃ ^{2 −} ions before stabilising ACC and calcite-like phases, while precluding the incorporation of Mg ²⁺ to avoid aragonite formation. Indeed, via Asp-Ca ²⁺ bridges, structure-less clusters coalescence into supersaturated protein-CaCO ₃ assemblies initiates calcite crystallisation. Therefore, Aspein plasticity, condensation susceptibility, and aspartic density critically modulate the pathway that defines CaCO ₃ polymorphism by inhibiting nucleation and controlling the growth of biogenic minerals. From a broader perspective, our study underpins some of the molecular and physico-chemical principles governing biomineralisation processes mediated by IDOMPs, the overlooked, yet prevalent, role of which was mechanistically unexplored.

Amyloids refer to a diverse group of highly ordered proteinaceous structures conserved across all of the domains of life that not only are involved in severe proteinopathies but also serve as unique functional platforms. They are notably organized around a cross-β sheet core arranged into a dry steric zipper. Identifying regions promoting such type of assembly requires investigating, usually at the atomistic level, their structural and morphological properties. Although more amyloid structures are being solved and described by means of high-resolution experimental techniques, most amyloidogenic systems remain experimentally elusive or uncharacterized. To overcome such limitations, we propose a multilevel sequence-based computational approach taking advantage of aggregation-oriented predictors and modelers coupled with all-atom equilibrium molecular dynamics (MD) simulations. As an example of this approach, we introduced our protein of interest, the intrinsically disordered zinc finger DPF3a, to the pipeline, thus identifying a hit hexapeptide (⁵²NCYIWM⁵⁷) exhibiting remarkable pro-amyloid features. Indeed, this peptide was shown to stabilize different steric zipper topologies, spontaneously self-assemble into β-sheeted oligomers, and to act as an amyloid seed enabling the conversion and addition of β-sheet multimers for fibril elongation, which echoes the pathological repertoire of DPF3a. In comparison, simulations performed on a peptide sequence consensually predicted as nonamyloid (³³AERSVR³⁸) congruently underscored its inability to maintain a steric zipper configuration and nucleate into oligomers enriched in β character. Using only the sequence of a target protein as starting input, our methodology noteworthily proved to be reliable for detecting aggregation-prone regions, assessing their amyloidogenicity, and elucidating their fibrillation mechanisms while holding promise for the design of new anti-amyloid drugs and amyloid-inspired biomaterials.

Fatty acid amide hydrolase (FAAH) inhibition holds therapeutic promise by enhancing endocannabinoid signaling. We previously identified β‐lactam compounds as reversible and selective h FAAH inhibitors with nanomolar potency. Here, we describe the synthesis of new β‐lactam derivatives to evaluate the effect of imide conformational constraints on activity and to eliminate potential metabolic soft spots. Bicyclic derivatives were designed to lock the imide in a syn configuration, but showed reduced potency compared with non‐cyclic analogs. Docking studies revealed that this weaker inhibition arises from an altered binding mode within the FAAH active site. In parallel, removal of ester and allyl groups did not affect inhibitory potency. Importantly, the optimized inhibitor enhanced N ‐acylethanolamine levels in J774 cells, supporting target engagement and suggesting improved metabolic stability. These results provide insights into the mode of action of β‐lactam FAAH inhibitors and guide the development of more potent, stable derivatives.

The double plant homeodomain fingers 3a (DPF3 isoform a) is a human epigenetic regulator involved in chromatin remodelling, cell division, and ciliogenesis. Most notably, this protein is deregulated in various cancer types and neurodegenerative diseases. In our previous work, the disorder nature of DPF3a, as well as its propensity to aggregate into amyloid fibrils, have been highlighted, making it an amyloidogenic intrinsically disordered protein (IDP). Due to their high chain accessibility, IDPs structure and function are modulated by phosphorylation. It has been reported that phosphorylation of DPF3a at S348 (pS348) by the casein kinase 2 (CK2) is implicated in cardiac hypertrophy. CK2 can also phosphorylate DPF3a at S138 (pS138), which is also located in an intrinsically disordered region (IDR). However, no structural information is available on phosphorylated DPF3a. In the present study, we investigated the effect of phosphorylation on DPF3a structural and aggregation properties. Two single-mutated phosphomimetics (S138E and S348E) were characterised in vitro and compared to DPF3a WT, while in silico analyses were performed on pS138 and pS348 to assess structural changes at the molecular level. Circular dichroism and fluorescence spectroscopy revealed that both phosphomimetics are hybrid IDPs, with increased turn and antiparallel β-sheet content as well as more buried aromatic residues compared to DPF3a WT, suggesting conformational rearrangements and a more folded N-terminal region. In silico characterisation supported these results, showing that phosphorylation of S138 and S348 induce extended conformation, especially the C-terminal extremity, due to electrostatic repulsion, while local folding occurs due to a proximity with arginine and lysine residues. Furthermore, spectroscopic and microscopic analyses unveiled that S138E and S348E exhibit slower fibrillation kinetics compared to DPF3a WT involving distinct aggregation mechanisms.