Venue: Centre Broca
Ivana Trivunovic
Team: Choquet (IINS) 
Thesis supervisor: Matthieu Sainlos
Title
Directed evolution and engineering of molecular mechanisms of proteins in synaptic transmission
Abstract
Over the last decades, technological advancements have allowed to generate a more accurate vision of the complex organization of neuronal cytoskeleton and synapses. These advancements span from significant development of different imaging techniques, as well as the molecular tools currently used to probe different cellular compartments and specific proteins in excitatory synapses. Despite these progresses, our understanding of a number of structural and functional elements of these specialized neuronal compartments are still incomplete. Previously developed tools, such as synthetic antibodies and fusion proteins, need further improvement to achieve precise structural and functional probing and modulation of endogenous proteins. The work of this thesis was devoted to exploiting protein engineering techniques to achieve highly diversified genomic libraries using site-directed mutagenesis and high-throughput screening. This allows for applying evolutional pressure on libraries containing billions of sequences to identify the best candidate binders based on the human 10th fibronectin domain. Intrabody library design and selections were focused on two cytoskeletal proteins, talins and spectrins, in charge of neuronal development and synaptic formation and stabilization. Talin plays a critical role in cellular communication with the extracellular matrix and maintaining the overall cellular stability and integrity. It contains two distinguishable functional domains, the FERM N-term and the talin rod domain. In the context of this work, we report three highly specific binders to talin rod-subdomains R1R2, R9 and R11. Complexes of these binders with their target domains have been characterized with 2D NMR, providing detailed insight into the target domains’ binding epitopes. Spectrins create a highly regular protein network with actin rings across the cell, spanning from the axon initial segment to dendritic spines. In this context, small domains of αII-spectrin and β(II-IV)-spectrins were the targets in our selections, resulting in several promising candidates for structural and functional probing of spectrins. Protein-protein interaction modulation was focused on inhibiting several proteins in the post- synaptic density region, involved in glutamate receptor trafficking, synapse stabilization and signal transduction from binding to the PDZ domains of PSD95. This approach in protein-protein inhibition of the PDZ domain will allow for the blocking of only one of many interactions of the PDZ domain and shed more light on the contribution of each PDZ domain-mediated interaction in the complex regulation of synaptic plasticity. Upon full characterization, these intrabodies and affinity clamps will provide a new set of genetically encoded tools for endogenous protein labelling and modulation.
Keywords: directed evolution, protein engineering, PPI inhibition, imaging probe design, phage display, synaptic transmission
Jury
- Nathalie Sans (University of Bordeaux)
- Arnaud Gautier (University of PSL)
- Gerti Beliu (University of Wuerburg)
- Matthieu Sainlos (University of Bordeaux)