The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel whose function is tightly linked to its conformational dynamics and is influenced by the composition of its membrane lipid environment. Despite high-resolution three-dimensional (3D) structures, the molecular determinants that stabilize specific CFTR conformations and enable ion conduction remain incompletely understood. Here, we performed all-atom molecular dynamics (MD) simulations of the human CFTR 3D structure in both the apo and VX-770 (ivacaftor)-bound states, embedded in a heterogeneous lipid bilayer, in order to systematically analyze electrostatic interactions, linking amino acids to each other as well as to anions and membrane lipids. We identified 557 electrostatic interactions between charged and polar amino acid side chains, which we systematically mapped across the CFTR 3D structure. They are organized into specific regions, with a subset showing high frequency and conservation across simulations, suggesting a structural role in stabilizing CFTR architecture. In contrast, more transient electrostatic interactions were detected in dynamic regions potentially linked to conformational transitions or other functional roles. Irregularities in transmembrane (TM) helices often incorporate amino acids involved in electrostatic interactions. Many basic and polar residues involved in electrostatic interactions also engaged in anion coordination, underscoring their contribution to ion conduction. In addition, some showed selective interactions with cholesterol and phosphatidylserine, revealing spatially organized lipid binding, particularly at the level of the lasso and in the vicinity of the VX-770 binding site, which may mark regions important for allosteric communication. VX-770 binding preserved the global architecture of the electrostatic interaction networks but induced subtle shifts, acting on specific salt bridges. Regardless of whether VX-770 is present or not, a secondary portal displayed between TM10/TM12 emerged from these MD simulations, in addition to the main TM4/TM6 portal, whose morphology and diameter are controlled by a fluctuating salt bridge. Two exit routes also appeared for the exit of anions toward the extracellular milieu. Altogether, our integrative analysis highlights how dynamic electrostatic networks, together with ion and lipid interactions, support CFTR's structural plasticity and functional modulation, offering molecular insights into potentiation mechanisms and into the specific evolution of CFTR in the ABC transporter superfamily.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe autosomal recessive cholestatic liver disease due to variations in ATP-binding cassette subfamily B member 11 (ABCB11) gene. The clinical and molecular consequences of two missense variations affecting the same ABCB11 residue (T463) were characterized, and pharmacological strategies were investigated. Clinical and genetic data were collected from two PFIC2 patients carrying p.T463I or p.T463P substitutions. A three-dimensional (3-D) structure analysis was performed to predict substitution impacts. ABCB11T463I and ABCB11T463P variants were expressed in HepG2 and Madin-Darby canine kidney cells to assess their subcellular localization and functional activity. Pharmacological modulators were tested to correct the defects. The patient carrying ABCB11T463I exhibited a mild phenotype and responded to surgical biliary diversion. Conversely, the patient carrying ABCB11T463P required a liver transplantation before age one. 3-D structure and in vitro analyses predicted a functional defect in both variants and a folding defect for the T463P variant. In vitro, ursodeoxycholic acid combined with glycerol phenylbutyrate increased ABCB11T463P canalicular expression (40.2 ± 7.7% of the wild type, P < 0.0001) and improved transport activity (32.4 ± 10.3% of the wild type, P < 0.0001). VX-770 and SBC040 increased ABCB11T463I function from 37.9 ± 2.5% (DMSO) to 73.2 ± 12.3% and 76.1 ± 17.5%, respectively, of the wild-type activity (P < 0.0001). ABCB11 missense variations, even affecting the same residue, can cause various molecular defects, resulting in mild to severe phenotypes. 3-D structure and in vitro analyses could be used to predict the severity of missense variants and guide the treatment of PFIC2 patients with pharmacological modulators.NEW & NOTEWORTHY Patients with progressive familial intrahepatic cholestasis type 2 (PFIC2) due to ATP-binding cassette subfamily B member 11 (ABCB11) missense variations may present various phenotypes and exhibit different response patterns to treatments. This study exemplifies this variability and provides an in silico and in vitro framework for characterizing and classifying ABCB11 missense variants. This classification could serve to guide treatment strategies, stratify patients with missense variants in clinical trials, and predict patient outcomes.

MD simulations of human CFTR (PDB 6O2P):Equilibrium MD simulations were performed on the ATP-bound CFTR structure, with and without the potentiator VX-770 (Ivacaftor). The protein is embedded in a heterogeneous lipid bilayer composed of POPC, POPE, POPS, cholesterol (CHL1), and the sphingolipid DSM, at an approximate 3:3:2:1:1 ratio. Simulations were run using the CHARMM36 force field and the GROMACS MD engine. Each folder contains the input files and trajectories required for analysis. Each simulation contains the input pdb file and the trajectorie (XTC/DCD) required for analysis (e.g, apo1.pdb and apo1.xtc). Processed analysis files (TSV/CSV files).These include residue side chain – side chain contacts (PS.tsv, QNPS.tsv), side chain – main chain contacts (PSmain.tsv, QNPSmain), residue–lipid contacts (Lipids.tsv, QN_Lipids.tsv) and residues-anion contacts (Anion.tsv, QN_Anion.tsv), and secondary-structure analysis (combined_secstr_8sim.csv, showing percentage of alpha helix along each MD). Python notebook used in the manuscript.The notebook analysis_fig_pub.ipynb contains the exact code used to generate the supplementary figures using the processed analysis files.

Some bacteria genetically control the biomineralisation of intracellular amorphous calcium carbonates (iACC) with potential implications for microbial physiology, evolution, bioremediation and biogeochemical cycling. Until now, this capacity has been documented in Cyanobacteria, the giant gammaproteobacterium Achromatium and a few magnetotactic Pseudomonadota and Nitrospirota. Here, we report the discovery of iACC biomineralisation in members of the Methylococcaceae, a family of aerobic methanotrophic Gammaproteobacteria. A homologue of the ccyA gene, a diagnostic marker for iACC formation in Cyanobacteria, was identified in several Methylococcaceae genomes, based on the conserved C-terminal (GlyZip)3 domain of the encoded calcyanin protein. Moreover, two cultivated strains, Methylococcus geothermalis and Methylococcus mesophilus, whose genomes contained the ccyA gene, were consistently shown to form iACC. The ccyA genes of Methylococcaceae and Microcystis share higher sequence similarity than with other Cyanobacteria, suggesting horizontal gene transfer (HGT) from an ancestral Microcystis-like cyanobacterium to Methylococcaceae. This finding extends the known taxonomic distribution of ccyA and suggests that the capability to biomineralize iACC was acquired by HGT. The discovery of iACC in methane-oxidising Methylococcaceae highlights a previously unrecognised coupling between calcium carbonate biomineralisation and methane cycling in aquatic environments, suggesting an overlooked role of iACC formation in microbial carbon storage and local geochemical regulation.

We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.

Here we show that somatic genetic rescue is frequent in telomere biology disorders (TBDs) caused by germline ZCCHC8 variants. Our results highlight the critical intrinsic role of ZCCHC8 in human hematopoiesis and a potential mechanism for disease modification in TBDs.

Oncostatin M (OSM) is a cytokine with the unique ability to interact with both the OSM receptor (OSMR) and the leukemia inhibitory factor receptor (LIFR). On the other hand, OSMR interacts with IL31RA to form the interleukin-31 receptor. This intricate network of cytokines and receptors makes it difficult to understand the specific function of OSM. While monoallelic loss-of-function (LoF) mutations in OSMR underlie autosomal dominant familial primary localized cutaneous amyloidosis, the in vivo consequences of human OSM deficiency have never been reported so far. Here, we identified 3 young individuals from a consanguineous family presenting with inherited severe bone marrow failure syndromes (IBMFS) characterized by profound anemia, thrombocytopenia, and neutropenia. Genetic analysis revealed a homozygous 1 base-pair insertion in the sequence of OSM associated with the disease. Structural and functional analyses showed that this variant causes a frameshift that replaces the C-terminal portion of OSM, which contains the FxxK motif that interacts with both OSMR and LIFR, with a neopeptide. The lack of detection and signaling of the mutant OSM suggests a LoF mutation. Analysis of zebrafish models further supported the role of the OSM/OSMR signaling in erythroid progenitor proliferation and neutrophil differentiation. Our study provides the previously uncharacterized and unexpectedly limited in vivo consequence of OSM deficiency in humans.

Cystic fibrosis (CF) care has been revolutionized by CFTR modulators, particularly the triple combination elexacaftor/tezacaftor/ivacaftor (ETI). However, a subset of people with CF (pwCF) carrying ETI-unresponsive variants still lack effective therapies. A nextgeneration modulator combination, vanzacaftor/tezacaftor/deutivacaftor (VTD), shows promise in addressing this gap. Phase 3 trials report superior efficacy of VTD in reducing sweat chloride and suggest potential benefit for 31 CFTR variants previously deemed ETI-unresponsive based on limited in vitro data. Reassessment of these variants reveals that some demonstrate residual function or borderline ETI responsiveness, challenging their unresponsive classification. Differences in molecular interactions, particularly within NBD1 and modulator-binding sites, may account for variant-specific responses. Patient-derived intestinal organoid (PDIO) assays showed significant functional improvement with VTD, but not ETI, in 2 pwCF with G458V and G85R mutations, supporting the notion of distinct mechanisms of action. These findings underscore the need to refine CFTR variant classifications and highlight the limitations of current in vitro thresholds. While PDIOs offer physiologically relevant insights, clinical outcomes remain the ultimate determinant of therapeutic benefit. Broader access to raw data and individualized in vitro-clinical correlations are essential for informed therapeutic decisions. VTD offers new hope for pwCF with rare or previously unresponsive variants, reinforcing the importance of a personalized, datadriven approach to CF care.

Hydrophobic Cluster Analysis (HCA) adds secondary structure information to the analysis of protein amino acid sequence. Focusing on the elementary building blocks of protein folds, this approach has proved to be a powerful tool for detecting distant (hidden) relationships between proteins. At a time when huge masses of data are now available, both in terms of protein sequences and models of three-dimensional structures, it still constitutes a relevant tool for analyzing structural features at the scale of whole proteomes, enabling, among other things, to characterize the continuum between disorder and order and to explore the characteristics of protein dark matter. The aim of this mini-review is to provide a brief overview of this approach, describing its principles and achievements, recent developments and future prospects.

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulator VX-445 (Elexacaftor) used to treat cystic fibrosis presents both corrector and potentiator activities. This drug binds to a pocket within the CFTR membrane-spanning domain (MSD) assembly, in contact with the lasso motif. We have previously shown that the corrector activity of VX-445 is modulated by mutations within MSD1 and the nucleotide binding domain NBD1. Here, we evaluate if mutations affecting VX-445's corrector activity also affect its potentiator activity. Responses to increasing concentrations of VX-445 were measured using the halide sensitive fluorescent assay after transfection of CFTR mutants in HEK293 cells. Results show that VX-445 potentiated gating mutants causing cystic fibrosis located in the NBDs (NBD1 G551D and NBD2 G1349D) and in the IntraCellular Loops (ICL1 G178R and ICL3 G970R). Mutations within the VX-445 binding site inhibited potentiation of G551D, contrary to some mutations located outside this site that affected its corrector activity, two of which (M212A and F224A) were found to induce CFTR gain-of-function. Potentiation of G551D was also observed with correctors VX-809 and VX-121. In conclusion, CFTR modulator VX-445 can promote channel activity of gating mutants, an effect which is dependent on the integrity of its binding site. Potentiation could also be observed with correctors VX-809 and VX-121, indicating that more generally, CFTR correctors can promote channel activity.

The hepato-canalicular adenosine triphosphate (ATP)-binding cassette transporter ABCB4/MDR3 is responsible of the secretion of phosphatidylcholine (PC) into bile. Variations in ABCB4 gene induce a spectrum of cholestatic liver diseases, the most severe form is progressive familial intrahepatic cholestasis type 3 (PFIC3). The purpose of our study was to investigate the impact and potential rescue of four ABCB4 missense variants identified in patients, two of which (T437I and T1077M) affect homologous amino acids in the Walker A motifs in the two nucleotide-binding sites NBS2 and NBS1, respectively and the other two (S242R and S346I) affect residues of the transmembrane helices 4 and 6, respectively. The functional role of the four amino acids was assessed by analysis of three-dimensional (3D) structures and molecular dynamics (MD) simulations in a lipid bilayer. For functional validation, the mutants were reproduced in a plasmid encoding the human ABCB4 protein. The localization, the processing and the PC secretion activity of the mutants were studied after transfection in cell models. As the wild-type ABCB4, all four mutants expressed and trafficked efficiently to the canalicular membrane of HepG2 cells, but were dramatically impaired in PC secretion activity. This behavior can be explained by the critical positions of the amino acids in the NBSs and in a region participating in the substrate transport. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiators ivacaftor and Small Binder of CFTR 219 (SBC219) significantly rescued the function defect of the mutants with differential sensitivity. Our results demonstrate the importance of the four mutated residues for ABCB4 function, which may explain the pathogenic phenotype. They provide an experimental evidence that targeted pharmacotherapy for genetic diseases caused by ABCB4 deficiency is likely to be mutation-specific.

Despite the biological and pharmaceutical importance of protein-protein interactions, accurately predicting dissociation free-energies and rates remains an outstanding challenge. Here, we predict these quantities from all-atom molecular dynamics simulations of ribonuclease barnase with its inhibitor barstar. We characterized the detailed contribution of different residues at the interface, along with the role of water, identifying three distinct binding pathways. Using Langevin models, we inferred the free-energy and diffusion landscape of the process, hence the kinetic properties, systematically evaluating the model's quality across different collective variables, and obtaining robust dissociation rate predictions. Our computational scheme, based on unbiased molecular dynamics trajectories initialized in the dissociated state, is significantly simpler than previously proposed approaches, and it is very data-efficient, requiring tens of sub-microsecond simulations. The results open the way to the systematic study of protein-protein interactions, including atomic-level details on the mechanism and kinetics that are paramount for biomedical applications.

In eutherians, the different elements of the insulin/insulin-like growth factor (IGF) system (ligands, receptors, binding proteins, proteases, inhibitors of proteases…) interact with each other. In the present work, we were interested in the question of when these elements appeared in the course of evolution and whether they appeared at the same evolutive node or one after the other.For this purpose, we have considered phylogenetic relationships extracted from two versions of Ensembl (releases 80 and 113). Moreover, we considered remote relationships detected by comparison of experimental or predicted 3D structures with 3D structures predicted at proteome scale, after validations by independent and reciprocal sequence similarity searches when needed.We showed that insulin-like/IGF peptides and their receptors appeared in non-vertebrate species, as well as the protein encoded by the non-vertebrate ecdysone-inducible gene L2 (Impl2), whereas IGF-binding proteins (IGFBPs) have appeared in vertebrate ancestor. Through structure and sesquence similarity searches, homologs of IGFBP-1/2/5, GH and GHR were detected in more distant species than thought to date (in Lamprey for − 1/2, Amphioxus for − 5, Lamprey for GH/GHR). We also showed that the pregnancy-associated plasma protein A (PAPP-A) and PAPP-A2 metzincin proteases, which degrade several IGFBPs, might have appeared in non-vertebrates and that their two inhibitors, proMBP and STC, probably appeared before both proteases. Overall, these results show that ancestors of insulin-like peptides appeared before the divergence of vertebrate and non-vertebrate species, as well as their receptors, and that the regulation strategies of this ligand might be different between both clades.

Novel dominant PSMB10 variants cause severe combined immunodeficiency and life-threatening liver disease in three pediatric patients. These mutations impair immunoproteasome assembly through a dominant-negative effect on PSMB9, likely disrupting lymphocyte survival and endothelial function, and are associated with poor outcomes after hematopoietic stem cell transplantation.

Background and objective: Despite significant advances, the molecular basis and thus patient-tailored therapeutic options for inborn errors of immunity remain unknown in a significant number of patients. Methods: We investigated three patients from unrelated families with inborn errors of immunity and severe liver disease. Clinical, immunological, and histological (only in one patient for the latter) data were collected, and trio whole exome sequencing was performed. Results: Whole exome sequencing identified heterozygous de novo variants in PSMB10 (p.Asp205Ala and p.Ser208Phe), a gene encoding a specific subunit of the immuno- and thymoproteasome. Analysis showed poor integration into the proteasome complex of PSMB10 variants, thereby exerting a dominant-negative effect on the PSMB9 subunit. Conclusion: Dominant PSMB10 variants should be considered in genetic screening for SCID and CID, especially in patients with associated liver disease. Very severe endothelial-like disease before and after HSCT and very poor outcomes after HSCT should weigh up the indication of HSCT.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ATP-gated anion channel whose function is tightly linked to its conformational dynamics and is influenced by the composition of its membrane lipid environment. Despite high-resolution three-dimensional (3D) structures, the molecular determinants that stabilize specific CFTR conformations and enable ion conduction remain incompletely understood. Here, we performed all-atom molecular dynamics (MD) simulations of the human CFTR 3D structure in both the apo and VX-770–bound states, embedded in a heterogeneous lipid bilayer, in order to systematically analyze electrostatic interactions, linking amino acids to each other as well as to anions and membrane lipids. We identified 558 electrostatic interactions between charged and polar amino acid side chains, which we systematically mapped across the CFTR 3D structure. They are organized into specific regions, with a subset showing high frequency and conservation across simulations, suggesting a structural role in stabilizing CFTR architecture. In contrast, more transient electrostatic interactions were detected in dynamic regions potentially linked to conformational transitions or other functional roles. Irregularities in transmembrane (TM) helices often incorporate amino acids involved in electrostatic interactions. Many basic and polar residues involved in electrostatic interactions also engaged in anion coordination, underscoring their contribution to ion conduction. In addition, some showed selective interactions with cholesterol and phosphatidylserine, revealing spatially organized lipid binding, particularly at the level of the lasso and in the vicinity of the VX-770 binding site, which may mark regions important for allosteric communication. VX-770 binding preserved the global architecture of the electrostatic interaction networks but induced subtle shifts, reinforcing specific salt bridges and enhancing anion contacts, especially around a portal displayed between TM10/TM12. This one is located opposite to the main TM4/TM6 portal, whose morphology and diameter is controlled by a fluctuating salt bridge. Regardless of whether VX-770 is present or not, two exit routes also emerged from these MD simulations. Altogether, our integrative analysis highlights how dynamic electrostatic networks, together with ion and lipid interactions support CFTR’s structural plasticity and functional modulation, offering molecular insights into potentiation mechanisms and into the specific evolution of CFTR in the ABC transporter superfamily. ### Competing Interest Statement The authors have declared no competing interest. * 3D : three-dimensional CHL : cholesterol CF : Cystic Fibrosis CFTR : Cystic Fibrosis Transmembrane Conductance Regulator cryo-EM : cryo-electron microscopy HB : Hydrogen Bond SB : Salt Bridge ECL : ExtraCellular Loop TMD : TransMembrane Domain ICL : IntraCellular Loop TM : TransMembrane NBD : Nucleotide Binding Domain MD : Molecular Dynamics DSM : sphingolipid POPC : 1-palmitoyl-2-oleoylphosphatidylcholine) POPE : 1-palmitoyl-2-oleoylphosphatidylethanolamine) POPS : 1-palmitoyl-2-oleoylphosphatidylserine. Vaincre la Mucoviscidose, https://ror.org/00gp9bw49

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic variations in the ABCB11 (ATP-binding cassette B11) gene encoding the canalicular bile salt export pump (BSEP). Some missense variants identified in patients with PFIC2 do not traffic properly to the canalicular membrane. However, 4-phenybutyrate (4-PB) has been shown in vitro to partially correct the mis-trafficking of selected variants, resulting in an improvement of the medical conditions of corresponding PFIC2 patients. Herein, we report the ability of 4-PB analogous or homologous drugs and of non-4-PB related chemical correctors to rescue the canalicular expression and the activity of the folding-defective Abcb11R1128C variant. New compounds, either identified by screening a chemical library or designed by structural homology with 4-PB (or its metabolites) and synthesized, were evaluated in vitro for their ability to (i) correct the canalicular localization of Abcb11R1128C after transfection in hepatocellular polarized cell lines; (ii) restore the 3H-taurocholate transport of the Abcb11R1128C protein in Madin-Darby canine kidney (MDCK) cells stably co-expressing Abcb11 and the sodium taurocholate co-transporting polypeptide (Ntcp/Slc10A1). Glycerol phenylbutyrate (GPB), phenylacetate (PA, the active metabolite of 4-PB), 3-hydroxy-2-methyl-4-phenylbutyrate (HMPB, a 4-PB metabolite analog chemically synthesized in our laboratory) and 4-oxo-1,2,3,4-tetrahydro-naphthalene-carboxylate (OTNC, from the chemical library screening) significantly increased the proportion of canalicular Abcb11R1128C protein. GPB, PA, ursodeoxycholic acid (UDCA), alone or in combination with 4-PB, suberoylanilide hydroxamic acid (SAHA), C18, VX-445, and/or VX-661, significantly corrected both the traffic and the activity of Abcb11R1128C. Such correctors could represent new pharmacological insights for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the transporter's traffic.

The Major Facilitator Superfamily (MFS) is the largest known family of secondary transporters. These proteins share a common architecture comprising two lobes, each including 6 transmembrane (TM) helices, related by twofold pseudosymmetry. They transport a wide range of substrates through large conformational changes relying on the opening and closing of gates located on either side of biological membranes. Human ferroportin 1 (HsFPN1), the sole characterized mammalian iron exporter, follows this pattern. It is, however, characterized by an unusual intracellular gate, formed by two asymmetric networks of non-covalent bonds linking the two lobes. We studied the behavior of these networks in all-atom molecular dynamics simulations and functionally assessed the effect of alanine substitutions on HsFPN1 plasma membrane expression and iron export activity. We identified two new critical residues, Arg156 and Tyr318, connecting the networks to each other and to one of two metal-coordinating sites, located in an unwound region of TM7. We extended the analysis to a previously unreported missense variation, p.Gln478Arg, which was found to have a very strong impact on one of the two inter-lobe connection networks, and to result in a significant HsFPN1 loss-of-function. This led us to present the p.Gln478Arg substitution as a new pathogenic variation causing ferroportin disease. Together, our results provide new insights into the structure and dynamics of the human FPN1 inner gate and its asymmetry, shedding light on its potential role in the mechanism of iron export while offering a framework to better understand previously unexplained clinical observations.

Phylogenetically diverse cyanobacteria biomineralize intracellular amorphous calcium carbonate (iACC) inclusions. This includes several genotypes of the Microcystis genus, a potentially toxic, bloom-forming cyanobacterium found worldwide in freshwater ecosystems. While we ignore the biological function of iACC and the molecular mechanisms driving their formation, this process may impact local geochemical cycles and/or be used for bioremediation strategies. Recently, a marker gene of this biomineralization pathway, named ccyA, was discovered. However, the function of the calcyanin protein encoded by ccyA remains unknown. Here, based on an RNA-Seq approach, we assess the expression of the ccyA gene in Microcystis aeruginosa PCC 7806 during a 24 h day/night cycle. The ccyA gene shows a clear day/night expression pattern with maximum transcript abundances during the second half of the night. This is consistent with the assumption that iACC biomineralization is related with photosynthesis and may therefore follow a day/night cycle as well. Moreover, several genes directly co-localized upstream and downstream of ccyA, on the same DNA strand show a similar expression pattern, including a cax gene encoding a calcium/proton exchanger and a gene encoding a protein with a domain also present in the N-terminal region of calcyanins in many iACC-forming cyanobacteria. This suggests that they all could be part of an operon, and may play a concerted role in iACC formation. Last, several other genes involved in carbon concentrating mechanisms and calcium transport show an expression pattern similar to that of ccyA. Overall, this study provides a list of candidate genes that may be involved in the biomineralization of iACC by cyanobacteria and whose role could be, in the future, analyzed by biochemistry and genetics approaches.

The ubiquitously expressed small GTPase Ras-related protein 1B ( RAP1B ) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. . We demonstrate that G12E and the previously described G12V and G60R were gain-of- function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient's hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.