Biology subjects

Cendron, L.

Publications and source records attributed to Cendron, L..

10 recordsLinked to original sources

bioRxiv2026

Structural conservation and expanded functionality of hyper-stable human serum albumin variants

Human serum albumin (hSA) is the most abundant protein in human plasma, and its pharmacological properties, such as long plasma half-life mediated by the neonatal Fc receptor (FcRn) and its ability to bind endogenous and exogenous molecules, make it attractive for biotechnological applications. Currently, most wild type (WT) SAs are derived from human or bovine serum or produced in yeast and mammalian cells. Although well established, these methods are costly, difficult to reproduce, and not environmentally sustainable. Building on a previous study to design highly mutated hSA sequences, we extend the validation through an in-depth analysis of three engineered hSA variants; hSA1, hSA2, and hSA3, containing 16, 25, or 73 amino acid substitutions, respectively. These variants were designed for enhanced solubility, stability, and expression in Escherichia coli. All three variants showed low- micromolar affinities for hFcRn at pH 5.5, and negligible binding at pH 7.4. In a human endothelial cell-based recycling assay (HERA), the engineered hSA variants were recycled by hFcRn to the same extent as hSA isolated from serum. Exploring the properties of canonical drug-binding sites, warfarin affinity was comparable to WT hSA, whereas ibuprofen binding differed. Complementary cytotoxicity assays on human macrophages confirmed negligible toxicity and biocompatibility. A cryo-electron microscopy structure of hSA3 revealed that, despite extensive engineering, the native heart-shape of hSA, folding of domains, and its open conformation were preserved. These findings validate the structural integrity and functional adaptability of engineered hSA variants, underscoring their potential as versatile, animal-free solutions for next-generation therapeutics and biotechnological applications.

biochemistry↗

bioRxiv2025

A symbiotic MLO gene regulates root development via RALF34-triggered Ca2+ signalling in Lotus japonicus

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes, including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent evidence shows that MLO proteins can act as Ca2+-permeable channels in response to Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca2+-mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus, focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in root cells colonized by arbuscular mycorrhizal (AM) fungi, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting even in the absence of AM fungal colonization and in a Ca2+-dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca2+ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca2+ signalling. Together, these findings identify LjMLO4 as a molecular hub between peptide signalling, Ca2+ transport and root system architecture, highlighting how MLO proteins integrate developmental, nutritional and symbiotic cues.

plant biology↗

bioRxiv2025

Harnessing photosynthetic ATP for whole-cell biocatalysis in the cyanobacterium Synechocystis

Photosynthetic organisms use sunlight to produce ATP and NADPH powering their metabolism. Harnessing these products for driving biocatalytic reactions would enable to develop clean and sustainable alternatives for chemical reactions. In this study, we present the first demonstration that ATP produced from the photosynthetic process, can fuel a biocatalytic transformation in the whole-cell configuration. This result was achieved by expressing in the cyanobacterium Synechocystis sp. PCC 6803 an ATP-dependent enzyme, the {gamma}-Glutamyl-MethylAmide Synthetase from Methylovorus mays No. 9 (MmGMAS). The expressed enzyme was able to drive in the transgenic strain the light-driven biosynthesis of L-theanine. Consumption of ATP by the recombinant MmGMAS was even beneficial under strong illumination, protecting the photosynthetic electron transport from photodamage. These findings demonstrate the possibility of using photosynthetic microorganisms like Synechocystis as potential platform for sunlight driven biotransformations with wide potential biocatalytic applications. In this perspective, we further present the tridimensional structure of MmGMAS, which explains its promiscuous in vivo activity and provides the basis for its rational evolution.

biochemistry↗

bioRxiv2025

Identification and characterization of a small-molecule inhibitor of the Pseudomonas aeruginosa SOS response

The SOS response is among the most conserved pathways that promote the acquisition of antibiotic resistance in bacteria. This study aimed to identify and characterize small-molecule inhibitors of the SOS response system in the opportunistic pathogen Pseudomonas aeruginosa. A library of 318 drug-like compounds was screened for inhibition of RecA-induced LexA autoproteolysis, a key step in SOS activation. One hit compound, 3-(2-sulfanylanilino)propanoic acid, showed dose-dependent inhibition with an IC50 in the mid-micromolar range. Differential scanning fluorimetry and isothermal titration calorimetry analysis revealed that A12 binds to both RecA and LexA with low micromolar affinity. Mass spectrometry analysis demonstrated that A12 covalently modifies RecA likely via condensation, while it forms a disulfide bond with Cys104 of LexA. Inhibition was diminished under reducing conditions, confirming disulfide formation is crucial for A12 activity. Importantly, A12 did not impair LexAs ability to bind SOS box DNA sequences, which is needed to keep the SOS genes repressed. While A12s potency requires optimization, it represents a promising scaffold for developing anti-SOS compounds targeting P. aeruginosa.

biochemistry↗

bioRxiv2024

Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

Macrocyclic peptides provide an attractive modality for drug development due to their ability to bind challenging targes, their small size, and amenability to powerful in vitro evolution techniques such as phage or mRNA display. While these technologies proved capable of generating and screening extremely large libraries and yielded ligands to already many targets, they often do not identify the best binders within a library due to the difficulty of monitoring performance and controlling selection pressure. Furthermore, only a small number of enriched ligands can typically be characterised due to the need of chemical peptide synthesis and purification prior to characterisation. In this work, we address these limitations by developing a yeast display-based strategy for the generation, screening and characterisation of structurally highly diverse disulfide-cyclised peptides. Analysis and sorting by quantitative flow cytometry enabled monitoring the performance of millions of individual macrocyclic peptides during the screening process and allowed us identifying macrocyclic peptide ligands with affinities in the low micromolar to high picomolar range against five highly diverse protein targets. X-ray analysis of a selected ligand in complex with its target revealed optimal shape complementarity, large interaction surface, constrained peptide backbones and multiple inter- and intra-molecular interactions, rationalising the high affinity and exquisite selectivity. The novel technology described here offers a facile, quantitative and cost-effective alternative to rapidly and efficiently generate and characterise fully genetically encoded macrocycle peptide ligands with sufficiently good binding properties to even therapeutically relevant targets.

biochemistry↗

bioRxiv2024

Snapshots of Pseudomonas aeruginosa SOS response activation complex reveal structural prerequisites for LexA engagement and cleavage

Antimicrobial resistance represents a major threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assays in vitro, we elucidated the molecular events governing the SOS response activation in P. aeruginosa, focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that let RecA trigger the autocleavage and inactivation of the LexA repressor. These results provide the groundwork for designing novel antimicrobial strategies and for exploring the potential translation of Escherichia coli-derived approaches, to address the health-threatening implications of bacterial infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/585941v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@2fe1e4org.highwire.dtl.DTLVardef@19741e1org.highwire.dtl.DTLVardef@1664ddborg.highwire.dtl.DTLVardef@18195cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Physcomitrium patens flavodiiron proteins form a redox-dependent heterocomplex.

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by exploiting electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero multimer protein complex, that has never been isolated and characterized so far. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo role and regulation.

plant biology↗

bioRxiv2023

Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy

Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin decorated mitochondria are selectively recruiting autophagy receptors,which are required to terminate the organelle via autophagy. In this work we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria, and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons, and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

cell biology↗

bioRxiv2020

PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

Mutations in SPG11, encoding spatacsin, constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal and axonal transport impairments. However, the upstream mechanisms that regulate spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of spatacsin. The interaction is modulated by Protein Kinase A (PKA)-dependent phosphorylation of spatacsin at Ser1955, which initiates spatacsin trafficking from the plasma membrane to the intracellular space. Our study provides novel insight in understanding spatacsin physio-pathological roles with mechanistic dissection of its associated pathways.

cell biology↗

bioRxiv2018

In Silico Identification and In Vitro Validation of Novel KPC-2 β-lactamase Inhibitors

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type {beta}-lactamases are often reported as resistant to available {beta}-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non {beta}-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-{beta}-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the {beta}-lactam antibiotic meropenem by four fold.

biochemistry↗