Biology subjects

Callebaut, I.

Publications and source records attributed to Callebaut, I..

12 recordsLinked to original sources

bioRxiv2025

Intracellular amorphous calcium carbonate biomineralization in methanotrophic gammaproteobacteria was acquired by horizontal gene transfer from cyanobacteria

Some bacteria genetically control the biomineralization 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 biomineralization in members of the Methylococcaceae, a family of aerobic methanotrophic Gammaproteobacteria. A homolog of the ccyA gene, previously considered a diagnostic marker for iACC formation in Cyanobacteria, was identified in several Methylococcaceae genomes, based on a search of the conserved C-terminal (GlyZip)3 domain of the encoded calcyanin protein, with a sequence coverage higher than 60% and an E-value lower than 1e-20. Moreover, two cultivated strains, Methylococcus geothermalis and Methylococcus mesophilus, whose genomes contained the ccyA gene, were consistently shown to form iACC granules. The ccyA genes of Methylococcaceae and Microcystis share higher sequence similarity (47%) than with other Cyanobacteria (around 30%) within their common (GlyZip)3 domain, 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, possibly in environments such as those close to the oxyclines of lakes, where Cyanobacteria and Methylococcaceae commonly co-exist. The discovery of iACC in methane-oxidizing Methylococcaceae highlights a previously unrecognized coupling between calcium carbonate biomineralization and methane cycling in aquatic environments, suggesting that iACC formation may play an overlooked role in microbial carbon storage and local geochemical regulation.

microbiology↗

bioRxiv2025

Mechanistic insights into CFTR function from molecular dynamics analysis of electrostatic interactions

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 is controlled by a fluctuating salt bridge. Two exit routes also appeared for the exit of anions towards the extracellular milieu. Altogether, our integrative analysis highlights how dynamic electrostatic networks, together with ion and lipid interactions, support CFTRs structural plasticity and functional modulation, offering molecular insights into potentiation mechanisms and into the specific evolution of CFTR in the ABC transporter superfamily. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/690003v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@9bbfdaorg.highwire.dtl.DTLVardef@15086a6org.highwire.dtl.DTLVardef@1b8397corg.highwire.dtl.DTLVardef@1c620ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Diel changes in the expression of a marker gene and candidate genes for intracellular amorphous CaCO3 biomineralization in Microcystis

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.

microbiology↗

bioRxiv2024

Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease.

SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on these conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366 and Arg371) that are located at the membrane-cytosol interface and consistently interact with POPC and POPE lipid molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward-facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid phosphates. This result into a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt-bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease.

molecular biology↗

bioRxiv2023

Cholesterol modulates the human FPN1 iron export function in plasma membrane liquid-ordered microdomains

Ferroportin 1 (FPN1) is the only known mammalian iron efflux transporter. This multi-pass membrane protein, which adopts the Major Facilitator Superfamily fold, is tightly controlled by serum hepcidin to assure maintenance of adequate cellular and systemic iron levels. Earlier studies have shown that cholesterol-lowering drugs can reduce FPN1 expression in liquid-ordered plasma membrane microdomains and its sensitivity to hepcidin. However, the molecular mechanism by which cholesterol depletion regulates the localization of FPN1 at the cell surface remains unknown. In biochemical experiments, we show that cholesterol depletion reduces the iron export function of FPN1. Repletion with cholesterol restores FPN1 activity. This is not observed with the diastereoisomer epicholesterol, suggesting a direct interaction between cholesterol and FPN1. Consistent with this, we demonstrate that mutants affecting the key tyrosine residues of three cholesterol-recognition amino acid consensus (CRAC/CARC) motifs have a negative impact on FPN1 activity, in manner that also decreases its abundance in ordered plasma membrane microdomains. A complementary structural analysis allows us to focus on a conserved CARC motif (CARC-1) located in a deep hydrophobic groove between transmembrane helices 1 and 5. Molecular docking suggests that this groove is well suited to cholesterol binding. All these findings indicate that the interaction between FPN1 and cholesterol is of major importance for the localization of FPN1 in ordered microdomains of the plasma membrane, which is necessary for its optimal activity, and so its responsiveness to hepcidin.

biochemistry↗

bioRxiv2023

Ku-binding motifs in RAG2, XLF, PAXX and MRI support functional redundancy during V(D)J recombination

The interaction of several partners with Ku through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Here, we first established more specifically the function of KBMs in V(D)J recombination as the molecular basis of functional redundancy between XLF and the NHEJ proteins MRI and PAXX. Then, given the functional redundancy between RAG2 and XLF, we explored the hypothesis of a KBM-mediated interaction between RAG2 and Ku. Through sequence alignment and biophysical methods, we identified a KBM at the C-terminus of RAG2 (R2CT) that mediates its interaction with Ku both in vitro and in cellulo. Notably, we showed that R2CT/Ku interaction is independent of the RAG nuclease activity. Finally, we demonstrated that the respective KBMs of RAG2 and XLF support their functional redundancy for V(D)J recombination.

molecular biology↗

bioRxiv2023

MiniBAR/KIAA0355 is a dual Rac and Rab effector required for ciliogenesis

Cilia protrude from the cell surface and play critical roles in in-tracellular signaling, environmental sensing and development. Actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1-and Rab35-binding protein with a truncated BAR domain that we named MiniBAR (aka KIAA0355/GARRE) which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles traffick-ing to the ciliary base and exhibits remarkable fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia resulting from abnormal Rac-GTP/Rho-GTP levels, increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hall-marks of ciliopathies including left-right asymmetry defects. Thus, MiniBAR is a unique dual Rac and Rab effector that con-trols both actin cytoskeleton and membrane trafficking for cili-ogenesis.

cell biology↗

bioRxiv2023

Toxoplasma Membrane Inositol Phospholipid Binding Protein TgREMIND Is Essential for Secretory Organelle Function and Host Infection

Apicomplexan parasites have specialized secretory organelles called rhoptries, micronemes, and dense granules that are essential for host infection. Here, we show that TgREMIND, a Toxoplasma gondii protein containing a membrane phospholipid interacting domain, is required for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain at the N-terminus, known to promote cell membrane bending, and a novel uncharacterized domain that we named REMIND for regulator of membrane interacting domain at the C-terminus. TgREMIND binds to PIP2 lipid species and both F-BAR and REMIND domains are necessary to ensure proper biological activities in vitro and in cellulo. Conditional depletion of TgREMIND results in the absence of dense granules and abnormal transparent rhoptries, leading to a severe inhibition of parasite motility, host invasion, and dissemination. Thus, our study demonstrates that TgREMIND is essential for the proper functioning of key secretory organelles required for successful infection by Toxoplasma.

cell biology↗

bioRxiv2021

A new gene family diagnostic for intracellular biomineralization of amorphous Ca-carbonates by cyanobacteria

Cyanobacteria have massively contributed to carbonate deposit formation over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein (calcyanin) family specifically associated with cyanobacterial iACC biomineralization. Calcyanin is composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologues. Calcyanin lacks detectable full-length homologs with known function. Yet, genetic and comparative genomic analyses suggest a possible involvement in Ca homeostasis, making this gene family a particularly interesting target for future functional studies. Whatever its function, this new gene family appears as a gene diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC. This significantly extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Phylogenetic analyses indicate that iACC biomineralization is ancient, with independent losses in various lineages and some HGT cases that resulted in the broad but patchy distribution of calcyanin across modern cyanobacteria. Overall, iACC biomineralization emerges as a new case of genetically controlled biomineralization in bacteria. Significance statementFew freshwater species of Cyanobacteria have been known to mineralize amorphous CaCO3 (ACC) intracellularly. Despite the geochemical interest of this biomineralization, its evolutionary history and molecular mechanism remain poorly known. Here, we report the discovery of a new gene family that has no homolog with known function, which proves to be a good diagnostic marker of this process. It allowed to find cyanobacteria in several phyla and environments such as seawater, where ACC biomineralization had not been reported before. Moreover, this gene is ancient and was independently lost in various lineages with some later horizontal transfers, resulting in a broad and patchy phylogenetic distribution in modern cyanobacteria.

evolutionary biology↗

bioRxiv2021

CFTR corrector efficacy is associated with occupancy of distinct binding sites

CFTR misfolding due to cystic fibrosis causing mutations can be corrected with small molecules designated as correctors. VX-809, an investigational corrector compound, is believed to bind CFTR directly to either the first membrane-spanning domain (MSD1) and/or the first nucleotide-binding domain (NBD1). Blind docking onto the 3D structures of these domains, followed by molecular dynamics (MD) simulations, revealed the presence of two potential VX-809 binding sites which, when mutated, abrogated corrector rescue. Mutations altering protein maturation are also shown to be not equally sensitive to the occupancy of the two sites by VX-809, with the most frequent mutation F508del requiring integrity of both sites and allosteric coupling with the F508del region while L206W only requires the integrity of the MSD1 site. A network of charged amino acids in the lasso Lh2 helix and the intracellular loops ICL1 and ICL4 is involved in the allostery between MSD1 and NBD1. Corrector VX-445, which is used in combination in clinics with VX-661, a structurally close analog of VX-809, to fully correct F508del, is also shown to occupy two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. In conclusion, VX-809 and VX-445 appear to bind different CFTR domains to alleviate specific folding defects. These results provide new insights into therapeutics understanding and may help the development of efficient corrector combinations.

molecular biology↗

bioRxiv2021

Intergenic ORFs as elementary structural modules of de novo gene birth and protein evolution

The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characterized the fold potential diversity of the amino acid sequences encoded by all intergenic ORFs (Open Reading Frames) of S. cerevisiae with the aim of (i) exploring whether the large structural diversity observed in proteomes is already present in noncoding sequences, and (ii) estimating the potential of the noncoding genome to produce novel protein bricks that can either give rise to novel genes or be integrated into pre-existing proteins, thus participating in protein structure diversity and evolution. We showed that amino acid sequences encoded by most yeast intergenic ORFs contain the elementary building blocks of protein structures. Moreover, they encompass the large structural diversity of canonical proteins with strikingly the majority predicted as foldable. Then, we investigated the early stages of de novo gene birth by identifying intergenic ORFs with a strong translation signal in ribosome profiling experiments and by reconstructing the ancestral sequences of 70 yeast de novo genes. This enabled us to highlight sequence and structural factors determining de novo gene emergence. Finally, we showed a strong correlation between the fold potential of de novo proteins and the one of their ancestral amino acid sequences, reflecting the relationship between the noncoding genome and the protein structure universe.

evolutionary biology↗

bioRxiv2018

HCAtk and pyHCA: A Toolkit and Python API for the Hydrophobic Cluster Analysis of Protein Sequences.

Motivation: Detecting protein domains sharing no similarity to known domains, as stored in domain databases, is a challenging problem, particularly for unannotated proteomes, domains emerged recently, fast diverging proteins or domains with intrinsically disordered regions.\n\nResults: We developed pyHCA and HCAtk, a python API and standalone tool gathering together improved versions of previously developed methodologies, with new functionalities. The developed tools can be either used from command line or from a python API.\n\nAvailability: HCAtk and pyHCA are available at https://github.com/T-B-F/pyHCA under the CeCILL-C license.

bioinformatics↗