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SUMMARY:Thesis defense - Anushka Nair
DESCRIPTION:Venue : Centre Broca \n\nAnushka Nair \nTeam : Structural Biology and Engineering of Neuronal Proteins\nIINS \nTitle\nNanobody discovery and affinity engineering approaches for targeting ionotropic glutamate receptors \nAbstract\nIonotropic glutamate receptors (iGluRs) are a family of ligand-gated ion channels which conduct most excitatory neurotransmission in the central nervous system. They are classified as; AMPA (GluA1-4)\, kainate (GluK1-5)\, NMDA (GluN1-3) and delta receptors (GluD1-2) and their subunits determine the ion selectivity and signaling properties. To understand the underlying mechanisms of excitatory transmission\, it is crucial to study iGluR composition\, localisation and trafficking at the synapse. My PhD project aims to discover high-affinity subunit-specific single-domain antibodies (Nanobodies) as versatile molecular tools to study the dynamic nature of these receptors. They will be used for super-resolution imaging to understand distribution and movement and for structural biology and proteomic studies to unravel native receptor conformations and compositions. \nIn order to discover such binders against the heteromeric GluK2/K5 kainate receptor\, I initially implemented the in vitro synthetic Nanobody (sybody) discovery workflow using three structurally distinct libraries (convex\, concave and loop) developed at the University of Zürich (Zimmermann et al.\, 2018). Each library\, having an initial diversity of 10^12-10^13 unique sequences\, was screened for binders by performing a combination of ribosome display and phage display (Zimmermann et al.\, 2020). The selected sybodies were biophysically and biochemically characterized but were shown to possess sub-optimal affinities and kinetic properties. In light of these results\, we opted for the conventional llama immunization approach to discover nanobodies against the GluK2/K5 receptor. \nBased on our previous observations\, we opted for an immune-derived nanobody library against the full-length GluN1/N2b NMDA receptor. Here\, we switched to yeast surface display coupled to selection via fluorescence activated cell sorting (FACS) (Chao et al.\, 2006). For this purpose\, I established an optimised and robust yeast display system using an in-house designed yeast display vector and the engineered yeast strain RJY100 (Van Deventer et al.\, 2015). Using this method\, we obtained a nanobody against the GluN1/N2b NMDAR\, which was then characterized to assess its binding properties. The resulting nanobody was identified as a moderately strong candidate\, needing further affinity engineering to improve its binding strength. \nFor this purpose\, we turned towards a continuous molecular evolution technique named AHEAD (Autonomous Hypermutation yEast surface Display) (Wellner et al.\, 2021). AHEAD is an in vitro affinity maturation platform which introduces mutations in a target sequence directly in yeast cells through an error-prone polymerase\, enabling continuous diversification and surface display of the protein repertoires. Building on results previously established in the lab\, I applied the AHEAD system to the interaction of Neuronal Pentraxin 1 with GluA4. This study had the dual purpose of enabling the further improvement of the NP1-GluA4 interaction as well as establishing the various parameters and conditions required for the implementation of the AHEAD system. Having done so\, the ideal next step is to successfully apply this continuous molecular evolution strategy for the NMDA receptor nanobody. \nAltogether\, my work describes synthetic nanobody library screening\, immune repertoire mining\, optimization of the yeast display strategy and affinity engineering of proteins\, thus establishing a versatile and powerful platform for generating nanobodies tailored for studying complex membrane proteins such as iGluRs. Notably\, the workflows I have established are compatible with a variety of molecular scaffolds which include natural\, synthetic and computationally designed repertoires. Moreover\, my PhD work leads to several future applications of these molecular tools in super-resolution microscopy\, structural biology\, proteomics and therapeutics\, thus contributing to functional and mechanistic studies of iGluRs. \nKeywords\nNanobodies\, neuronal receptors\, molecular neuroscience\, affinity engineering approaches \nPublications\n\nFeringa\, F.M.\, van Berkel\, A.A.\, NAIR\, A. and Verhage\, M.\, 2023. An atypical\, staged cell death pathway induced by depletion of SNARE-proteins MUNC18-1 or syntaxin-1. Journal of Neuroscience\, 43(3)\, pp.347-358. DOI: https://doi.org/10.1523/JNEUROSCI.0611-22.2022\nPeter Rout\, D.\, NAIR\, A.\, Gupta\, A. and Kumar\, P.\, 2019. Epidermolytic hyperkeratosis: clinical update. Clinical\, Cosmetic and Investigational Dermatology\, pp.333-344. (Review article). DOI: 10.2147/CCID.S166849\n\nJury\nSébastien FRIBOURG – Directeur de recherche\, INSERM\nRob MEIJERS – Head of Program\, Institute for Protein Innovation\nMiriam STOEBER – Associate Professor\, University of Geneva\nPascale MARCHOT – Directrice de recherche\, CNRS\nDerrick ROBINSON – Directeur de recherche\, CNRS\nJonathan ELEGHEERT – Chargé de recherche\, CNRS : Directeur de Thèse \n
URL:https://www.bordeaux-neurocampus.fr/en/event/thesis-defense-anushka-nair/
CATEGORIES:Thesis
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