Antonino Caliò

Bacterial microcompartments (BMCs) are self-assembling organelles that colocalize enzymes and metabolites of various biochemical pathways. Their selectively permeable shell is made entirely of protein, and the inherent properties of these systems make them attractive templates for bioengineering synthetic nanoreactors. Understanding the extent of shell stability in an extended range of conditions broadens the scope of chemistries that can be confined in these nanocompartments. Here we analyze the stability of in vitro assembled BMC shells and find that they retain their structure at high concentrations of various solvents and detergents, at temperatures up to 75 °C, and across a pH range from 3.9 to 11. Shell disassembly is observed below pH 3.9, but shells can be reassembled upon increasing the pH back to 8. These results expand the range of reactions that can be performed inside protein-based nanoreactors and underscore the potential of modular BMC shell proteins as versatile biomaterials.

Lipid nanoparticles (LNPs) have great potential as nucleic acid delivery vehicles; however, they trigger the production of inflammatory cytokines, which limits their medical applications. Developing non-inflammatory LNPs is challenging because the LNP’s ionizable lipid and the process of endosomal disruption are the major sources of LNP toxicity but are also essential for delivering nucleic acids. Here we demonstrate that ionizable lipids containing a carboxylic acid and an amine (termed S-lipid) switch their charged state between the pHs of 7.4 and 4.0, allowing them to generate LNPs (termed switchable nanoparticles) that efficiently encapsulate nucleic acid and trigger endosomal release without activation of the TLR4, complement, galectin-8 and platelet activating factor signalling pathways. Finally, we demonstrate that switchable nanoparticles are better at treating lipopolysaccharide-induced acute lung injury than traditional LNPs because they do not exacerbate pre-existing inflammation. Collectively, these results demonstrate that negatively charged ionizable lipids can mitigate the toxicity of LNPs.

The biological small-angle X-ray scattering (bioSAXS) beamline BM29 at the ESRF, operated by the ESRF–EMBL Joint Structural Biology and bioImaging Group (JSBIG), resumed user operation in September 2020 following the ESRF extremely brilliant source (EBS) upgrade. To exploit the high quality of X-ray beam delivered by this new fourth-generation synchrotron source, BM29 underwent significant refurbishment, including source optimization, instrument upgrades, and a complete redesign of the whole experimental control system. Here, we introduce the BioSAXS Customized Beamline Environment ( BSxCuBE-Web ), a new open-source, web-based platform designed to streamline, automate and enhance bioSAXS data collection. BSxCuBE-Web offers an intuitive and user-friendly interface for expert and non-expert users alike, to easily define and run bioSAXS experiments, as well as to monitor both raw and processed data. Its adaptable architecture facilitates its deployment on beamlines beyond the ESRF. In 2025 alone, BSxCuBE-Web supported more than 97 experiments involving over 256 users at BM29. The interface has been successfully validated using a wide range of biological samples and continues to be actively developed in response to bioSAXS user feedback and evolving experimental requirements. User experience reports have been overwhelmingly positive, highlighting significant improvements in efficiency, accessibility, and reliability during data collection.

Bases moléculaires de l'adaptation du protéome à haute pression dans les Archées extrémophiles: Les environnements extrêmes sont vastes et très répandus sur notre planète. Ils abritent une myriade de formes de vie adaptées. Par exemple, les habitats à Haute Pression Hydrostatique (HPH) englobent près de 60% de la surface de la Terre, alors que l'on retrouve des sources hydrothermales du fond du plus profond océan jusqu'aux hauteurs des montagnes. Comprendre comment les organismes extrémophiles se sont adaptés à ces conditions de vie est une question d'intérêt fondamental indéniable. C'est aussi une question pertinente lorsque l'on considère les origines de la vie sur Terre sachant que les sources hydrothermales des océans profonds, où sont présentes haute température et haute pression, ont été proposées comme le lieu possible de l'origine de la vie terrestre, à proximité d'une source d'énergie et à l'abri des radiations nocives émises par le jeune soleil. Cependant, jusqu'à aujourd'hui, les stratégies d'adaptation adoptées par les organismes adaptés aux HPH (piézophiles) restent encore méconnues. Les protéines sont la machinerie des cellules, et il faut que leur fonction soit maintenue optimale sous HPH afin que l'organisme survive. Bien que des études génomiques aient été tentées pour trouver un modèle général d'adaptation des protéines aux conditions de HPH, elles n'ont pas identifié de motif de substitution associé à l'adaptation à la HPH, alors que dans le même temps, les études sur les cellules entières ont prouvé l'existence d'une telle adaptation au niveau du protéome global. C'est pourquoi j'ai entrepris d'étudier des protéines orthologues isolées de deux Archées hyperthermophiles quasi-isogéniques, Thermococcus barophilus et Thermococcus kodakarensis, appartenant à l'ordre des Thermococcales, afin de mettre en évidence les bases physiques de l'adaptation à la HPH. Le choix de ces deux organismes, qui ne diffèrent que par leur pression de croissance optimale, garantit que les différences constatées sont liées à leur différence d'adaptation à la HPH. J’ai utilisé la diffusion neutronique, la spectroscopie de résonance magnétique nucléaire, la cristallographie à rayons X et les simulations de dynamique moléculaire pour effectuer une caractérisation structurelle et dynamique approfondie des Phosphomannose Isomérases et des protéines ribosomales S24e de ces deux organismes. Nos résultats montrent que les substitutions conservatives utilisant des résidus plus encombrants à des endroits clés du cœur hydrophobe améliorent considérablement la stabilité structurelle et dynamique des protéines piézophiles, sans affecter leur voie de repliement. De plus, une disposition différente de leurs résidus polaires et chargés à la surface entraîne une interaction réduite avec l'eau d'hydratation, ce qui leur permet de conserver leur flexibilité sous pression. Ces résultats sont en accord avec les études précédentes sur les cellules entières, et les développent de manière très détaillée, en identifiant la stratégie d'adaptation au niveau moléculaire. Cela constituera la base sur laquelle un modèle expliquant l'adaptation à la HPH au niveau génomique pourra être construit.

The extent of the effect of cellular crowding and cosolutes on the functioning of proteins and cells is manifold and includes the stabilization of the biomolecular systems, the excluded volume effect, and the modulation of molecular dynamics. Simultaneously, it is becoming increasingly clear how important it is to take the environment into account if we are to shed light on biological function under various external conditions. Many biosystems thrive under extreme conditions, including the deep sea and subseafloor crust, and can take advantage of some of the effects of crowding. These relationships have been studied in recent years using various biophysical techniques, including neutron and X-ray scattering, calorimetry, FTIR, UV-vis and fluorescence spectroscopies. Combining knowledge of the structure and conformational dynamics of biomolecules under extreme conditions, such as temperature, high hydrostatic pressure, and high salinity, we highlight the importance of considering all results in the context of the environment. Here we discuss crowding and cosolute effects on proteins, nucleic acids, membranes, and live cells and explain how it is possible to experimentally separate crowding-induced effects from other influences. Such findings will contribute to a better understanding of the homeoviscous adaptation of organisms and the limits of life in general.

Apolar lipids within the membranes of archaea are thought to play a role in membrane regulation.

Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known. Here, we show the molecular bases of these mechanisms through a joint structural and dynamical study of two orthologous proteins. We observed that pressure adaptation involves the decoupling of protein–water dynamics and the elimination of cavities in the protein core. This is achieved by rearranging the charged residues on the protein surface and using bulkier hydrophobic residues in the core. These findings will be the starting point in the search for a complete genomic model explaining high-pressure adaptation.

Different molecular motions within protomembranes probed by quasi-elastic neutron scattering.