Biology subjects

Vicogne, J.

Publications and source records attributed to Vicogne, J..

6 recordsLinked to original sources

bioRxiv2026

Total synthesis and structural characterization of a novel protein scaffold from the snail Biomphalaria glabrata.

Disulfide-rich miniproteins constitute compact and highly stable scaffolds of growing interest for molecular and structural engineering. Schistosomins are [~]80-residue proteins conserved across gastropods that form a long-standing orphan family whose structure and biological roles have remained unknown. Here, we report the total chemical synthesis and structural characterization of a schistosomin isoform from Biomphalaria glabrata, a medically relevant intermediate host of the parasite Schistosoma mansoni. Using state-of-the-art solid-phase peptide synthesis, chemoselective peptide ligation, and controlled oxidative folding, we obtained homogeneous well folded schistosomin suitable for biophysical and structural studies. High-resolution X-ray crystallography reveals a previously undescribed disulfide-rich fold defining a new class of miniprotein scaffold. Nano differential scanning fluorimetry and circular dichroism experiments demonstrate the remarkable thermal stability of this scaffold, while molecular dynamics simulations confirm the intrinsic rigidity of its disulfide-stabilized core and show that the two naturally occurring isoforms differing by a single residue exhibit nearly indistinguishable structural and dynamic properties. Finally, transcript and protein analyses across snail tissues provide the first spatial expression map of schistosomin in a medically relevant mollusk. Together, this work establishes schistosomin as a novel and robust miniprotein scaffold and provides a structural and biological framework for exploring its function and potential applications.

biochemistry↗

bioRxiv2024

Putative EGF ligand and receptor of Echinococcus multilocularis that are critical for parasite development

The neglected zoonosis alveolar echinococcosis (AE) is caused by infiltrative growth of the metacestode larval stage of the cestode Echinococcus multilocularis within host organs. We previously demonstrated that metacestode growth depends on the mitotic activity of a population of parasite stem cells, called germinative cells, but it is not yet clear which molecular mechanisms govern Echinococcus stem cell dynamics such as cell-cycle progression, self-renewal and differentiation. Based on previous reports showing that epidermal growth factor (EGF) signalling contributes to Echinococcus stem cell regulation, we herein characterized three EGF receptors of the parasite and demonstrated by RNAi and inhibitor assays that one of these, EmER1, is crucial for the development of metacestode vesicles from parasite stem cells. We also showed that EmER1 serves as a target for afatinib, an EGF receptor inhibitor with profound anti-parasitic activities in vitro and in vivo. By bioinformatic analyses and membrane-bound yeast two-hybrid assays, we identified a parasite-derived, neuregulin-like cognate ligand for EmER1, EmNRG, the expression of which is strongly upregulated in metacestode vesicles during clonal expansion of germinative cells. Furthermore, we demonstrate that RNAi knockdown of the EmNRG encoding gene drastically affects the ability of germinative cells to produce metacestode vesicles. We propose that EmNRG and EmER1 form a cognate ligand-receptor system utilized by E. multilocularis to regulate asymmetric versus symmetric division decisions of stem cells. These data are relevant for further studies into Echinococcus stem cell dynamics and for the development of EGF signalling-based anti-infectives against echinococcosis.

microbiology↗

bioRxiv2023

Optimized protocols for RNA interference in Macrostomum lignano

Macrostomum lignano, a marine free-living flatworm, has emerged as a potent invertebrate model in developmental biology for studying stem cells, germline, and regeneration processes. In recent years, many tools have been developed to manipulate this worm and to facilitate genetic modification. RNA interference is currently the most accessible and direct technique to investigate gene functions. It is obtained by soaking worms in artificial seawater containing dsRNA targeting the gene of interest. Although easy to perform, the original protocol calls for daily exchange of dsRNA solutions, usually until phenotypes are observed, which is both time- and cost-consuming. In this work, we have evaluated alternative dsRNA delivery techniques, such as electroporation and osmotic shock, to facilitate the experiments with improved time and cost efficiency. During our investigation to optimize RNAi, we demonstrated that, in the absence of diatoms, regular single soaking in artificial seawater containing dsRNA directly produced in bacteria or synthesized in vitro is, in most cases, sufficient to induce a potent gene knockdown for several days with a single soaking step. Therefore, this new and highly simplified method allows a very significant reduction of dsRNA consumption and lab work. In addition, it enables performing experiments on a larger number of worms at minimal cost.

developmental biology↗

bioRxiv2022

A Self-Purifying Microfluidic System for Identifying Drugs Acting Against Adult Schistosomes

The discovery of novel antihelminthic molecules to combat the development and spread of schistosomiasis, a disease caused by several Schistosoma flatworm species, mobilizes significant research efforts worldwide. In the absence of reliable and practical biochemical assays for measuring the viability of adult worms, the antischistosomicidal activity of molecules is usually evaluated by a detailed microscopic observation of worm mobility and/or integrity upon drug exposure. These assays have the disadvantage of being inacurate, subjective, biased by the limited in vitro worm viability and difficult to integrate at high density. We describe here a self-purifiying microfluidic system enabling the selection of healthy adult worms and the identification of molecules acting on the parasite. The worms are assayed in a dynamic environment that eliminates unhealthy worms that cannot attach firmly to the chip walls prior to being exposed to the drug. The detachment of the worms is also used as second step readout for identifying active compounds. We have validated this new fluidic screening approach using the two major antihelmintic drugs, Praziquantel and Artemisinin. The reported dynamic system is simple to produce and to parallelize. Importantly, it enables a quick, sensitive and reliable detection of antischistosomal compounds in no more than one day. This system can potentially be modified in the future to better mimic the natural habitat of the parasite.

bioengineering↗

bioRxiv2021

Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni

BACKGROUNDSchistosoma mansoni histone deacetylase 8 (SmHDAC8) is a privileged target for drug discovery. Invalidation of its transcription by RNAi leads to impaired survival of the worms in infected mice and its inhibition causes cell apoptosis and death. To determine why it is a promising therapeutic target the study of the currently unknown cellular signaling pathways involving this enzyme is essential. Protein partners of SmHDAC8 have been identified by yeast two-hybrid (Y2H) cDNA library screening and by mass spectrometry (MS) analysis. Among these partners we characterized SmRho1, the schistosome orthologue of human RhoA GTPase, which is involved in the regulation of the cytoskeleton. In this work, we validated the interaction between SmHDAC8 and SmRho1 and explored the role of the lysine deacetylase in cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGSWe characterized two isoforms of SmRho1, SmRho1.1 and SmRho1.2. Co-IP/Mass Spectrometry analysis identified SmRho1 partner proteins and we used two heterologous expression systems (Y2H assay and Xenopus laevis oocytes) to study interactions between SmHDAC8 and SmRho1 isoforms. To confirm SmHDAC8 and SmRho interaction in adult worms and schistosomula, we performed co-immunoprecipitation (Co-IP) experiments and additionally demonstrated SmRho1 acetylation using a Nano LC-MS/MS approach. A major impact of SmHDAC8 in cytoskeleton organization was documented by treating adult worms and schistosomula with a selective SmHDAC8 inhibitor or using RNAi followed by confocal microscopy. CONCLUSIONS/SIGNIFICANCEOur results suggest that SmHDAC8 is involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform. A specific interaction between SmHDAC8 and the C-terminal moiety of this isoform was demonstrated, and we showed that SmRho1 is acetylated on lysine K136. SmHDAC8 inhibition or knockdown using RNAi caused massive disruption of schistosomula actin cytoskeleton. A specific interaction between SmRho1.2 and SmDia suggested the existence of two signaling pathways that could regulate cytoskeleton organization via the two SmRho1 isoforms. Author summarySchistosoma mansoni is the major parasitic platyhelminth species causing intestinal schistosomiasis, for which around 200 million people are in need of treatment. Currently one drug, praziquantel, is the treatment of choice and its use in mass treatment programs, rendered imperative the development of new therapeutic agents. As new potential targets, we have focused on lysine deacetylases, and in particular Schistosoma mansoni histone deacetylase 8 (SmHDAC8). Previous studies showed that invalidation of the transcription of SmHDAC8 by RNAi led to the impaired survival of the worms after the infection of mice. The analysis of the 3D structure of SmHDAC8 by X-ray crystallography showed that the catalytic domain structure diverges significantly from that of human HDAC8 and this was exploited to develop novel anti-schistosomal drugs. Biological roles of SmHDAC8 are unknown. For this reason, we previously characterized its protein partners and identified the schistosome orthologue of the human RhoA GTPase, suggesting the involvement of SmHDAC8 in the modulation of cytoskeleton organization. Here, we investigated the interaction between SmHDAC8 and SmRho1 and identified two SmRho1 isoforms (SmRho1.1 and SmRho1.2). Our study showed that SmHDAC8 is indeed involved in schistosome cytoskeleton organization.

molecular biology↗

bioRxiv2020

A minimal hepatocyte growth factor mimic acting as a powerful agonist of the MET receptor tyrosine kinase for regeneration of epithelial tissues and organs.

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent on MET activation.

bioengineering↗