Biology subjects

Hanoulle, X.

Publications and source records attributed to Hanoulle, X..

4 recordsLinked to original sources

bioRxiv2026

A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

microbiology↗

bioRxiv2023

Strategy of selection and optimization of single domain antibodies targeting the PHF6 linear peptide within the Tau intrinsically disordered protein

The use of VHHs (Variable domain of the Heavy-chain of the Heavy-chain-only antibodies) as disease-modifying biomolecules in neurodegenerative disorders holds promises including to target aggregation-sensitive proteins. Exploitation of their clinical values dependents however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the Tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using nuclear magnetic resonance spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures of three complexes consisting of an optimized VHH and a peptide containing the Tau epitope. Interestingly, the ability of the VHH variants to inhibit Tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in our capacity to propose two VHH variants derived from the parent Z70 for their next development steps.

biochemistry↗

bioRxiv2021

Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

Tau proteins aggregate into filaments in brain cells in Alzheimers disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils. This lead VHH was optimized to improve its biochemical properties and to act in the intracellular compartment, resulting in VHH Z70. VHH Z70 was more efficient than the lead to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in a cellular model of Tau seeding also decreased the fluorescence-reported aggregation. Finally, intracellular expression of VHH Z70 in the brain of an established tauopathy mouse seeding model demonstrated its capacity to mitigate accumulation of pathological Tau. VHH Z70, by targeting Tau inside brain neurons, where most of the pathological Tau resides, provides a new tool to explore the optimal strategies of immunotherapy in tauopathies.

biochemistry↗

bioRxiv2019

Alzheimer’s genetic risk factor FERMT2 (Kindlin-2) is regulated by risk allele-binding of miR-4504 and leads to synaptic dysfunction in an APP-dependent manner

Although APP metabolism is being intensively investigated, a large fraction of its modulators are yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimers disease (AD), as a potential key modulator of axon guidance; a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism and that FERMT2 under-expression impacts axonal growth, synaptic connectivity and long-term potentiation in an APP-dependent manner. Lastly, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3UTR of FERMT2, induced a down-regulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 under-expression in neurons and insight on how this may influence AD pathogenesis.

pathology↗