Bruno Franzetti

M42 peptidases (TET) are ubiquitous, giant self-compartmentalized aminopeptidases forming unique tetrahedral structures. TETs may participate in protein homeostasis and amino acid recycling by processing peptides downstream of the proteasome and other related proteolytic complexes. Currently, only five TETs have been extensively studied in Archaea from the Thermococcales order, with a predominant focus on structural characterization. Therefore, a clear view of the diversity and biological activities of this large enzymatic family is lacking. In this study, we establish robust criteria for high-throughput identification of TET peptidases from sequence data and reveal their wide taxonomic distribution in Archaea. We functionally characterize six additional TETs, including four from Asgardarcheota. Our results identify two main functional classes, with generalist TETs displaying broad-spectrum activities, and specialized TETs with more selective activities. We also report the first description of methionyl- and isoleucyl-aminopeptidases in the TET family. In addition to their diverse substrate specificities, the characterized TETs exhibit distinct activation profiles, varying in optimal temperature, pH, and metal cofactor requirements. Combining functional and phylogenetic analyses, we propose a classification of TET peptidases into 11 groups. Notably, we identify generalist group 11 as the ancestral TET in Archaea, from which specialized TETs arose following gene duplication or horizontal transfer. Finally, we highlight the presence of multiple TETs with selective activities in heterotrophic and mixotrophic organisms, suggesting a metabolic role for these enzymes in the degradation of environmental peptides. Overall, our work illuminates an underexplored diversity of TET enzymes, uncovering a complex evolutionary history that shaped their functional diversification

While biophysical studies have unravelled properties of specific proteins in vitro , characterizing globally their native state within the cell remains a challenge. In particular, protein adaptation to harsh intracellular physical and chemical conditions is poorly understood. Extremophiles, which thrive in severe environments, are good models for the study of such adaptation. Five haloarchaeal species, isolated from hypersaline environments, were used to assess correlations between intracellular salt concentrations and molecular dynamics properties. In cellulo protein stability was measured using nano differential scanning fluorimetry, and neutron spectrometry was used to determine molecular dynamics resilience and global flexibility. It was found that high intracellular accumulation of Mg ²⁺ and low intracellular accumulation of K ⁺ were correlated with higher stability and resilience. Sequence traits associated with mean proteome halophilicity, such as decreased hydrophobicity and increased acidity, weighted by the relative abundance of each protein, were also correlated with stability and resilience. Haloferax mediterranei , however, was found to be an exception as its proteome showed the highest in cellulo molecular stability and resilience associated with fewest sequence traits related to halophilicity, highlighting the significance of the intracellular salt environment in determining proteome biophysical properties.

M42 peptidases are half-megadalton aminopeptidases characterized by a tetrahedral architecture (TET) ubiquitous across all domains of life. Despite their widespread occurrence, their evolutionary history and functional diversity remain largely unexplored. Here we show an unsuspected and largely untapped wealth of archaeal TET peptidases, exhibiting remarkable functional heterogeneity, as illustrated by the characterization of six novel enzymes. Using structural biology, phylogeny, and enzymatic studies, we establish robust criteria for high-throughput identification of TET peptidases and perform the first systematic study of their genomic distribution and functional diversity across the archaeal kingdom. We propose an 11-group classification for these enzymes and identify one group as the ancestral lineage that first emerged in Archaea. By coupling taxonomic distribution patterns with functional insights, we highlight the presence of multiple TET enzymes with selective activities in heterotrophic and mixotrophic organisms, suggesting a role for TET peptidases in the degradation of environmental peptides. Overall, this work illuminates the underexplored diversity of TET enzymes, uncovering a complex evolutionary history linked to their potential biological function.

Evidence of stable liquid water oceans beneath the ice crust of moons within the Solar System is of great interest for astrobiology. In particular, subglacial oceans may present hydrothermal processes in their abysses, similarly to terrestrial hydrothermal vents. Therefore, terrestrial extremophilic deep life can be considered a model for putative icy moon extraterrestrial life. However, the comparison between putative extraterrestrial abysses and their terrestrial counterparts suffers from a potentially determinant difference. Indeed, some icy moons oceans may be so deep that the hydrostatic pressure would exceed the maximal pressure at which hydrothermal vent organisms have been isolated. While terrestrial microorganisms that are able to survive in such conditions are known, the effect of high pressure on fundamental biochemical processes is still unclear. In this study, the effects of high hydrostatic pressure on DNA synthesis catalyzed by DNA polymerases are investigated for the first time. The effect on both strand displacement and primer extension activities is measured, and pressure tolerance is compared between enzymes of various thermophilic organisms isolated at different depths.

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl ₂ , CaCl ₂ and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui , Halobacterium salinarum , Haloferax mediterranei , Halorubrum sodomense and Haloferax volcanii . These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl ₂ for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein‐like biomarkers in extraterrestrial briny environments.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a beta-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.

The recent discovery of extrasolar Earth-like planets that orbit in their habitable zone of their system, and the latest clues of the presence of liquid water in the subsurface of Mars and in the subglacial ocean of Jupiter's and Saturn's moons, has reopened debates about habitability and limits of life. Although liquid water, widely accepted as an absolute requirement for terrestrial life, may be present in other bodies of the solar system or elsewhere, physical and chemical conditions, such as temperature, pressure, and salinity, may limit this habitability. However, extremophilic microorganisms found in various extreme terrestrial environments are adapted to thrive in permanently extreme ranges of physicochemical conditions. This review first describes promising environments for life in the Solar System and the microorganisms that inhabit similar environments on the Earth. The effects of extreme temperatures, salt, and hydrostatic pressure conditions on biomolecules will be explained in some detail, and recent advances in understanding biophysical and structural adaptation strategies allowing microorganisms to cope with extreme physicochemical conditions are reviewed to discuss promising environments for life in the Solar System in terms of habitability.

Adaption to environmental conditions is reflected by protein adaptation. In particular, proteins of extremophiles display distinctive traits ensuring functional, structural and dynamical properties under permanently extreme physical and chemical conditions. While it has mostly been studied with approaches focusing on specific proteins, biophysical approaches have also confirmed this link between environmental and protein adaptation at the more complex and diverse scale of the proteome. However, studies of this type remain challenging and often require large amounts of biological material. We report here the use of nanoDSF as a tool to study proteome stability and solubility in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results show that, as with single halophilic protein studies, proteome stability was correlated to the concentration of NaCl or KCl under which the cells were lysed and hence the proteome exposed. This work highlights that adaptation to environmental conditions can be experimentally observed at the scale of the proteome. Still, we show that the biochemical properties of single halophilic proteins can only be partially extrapolated to the whole proteome.