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Structural studies of the Roundabout protein family

Abstract : Neuronal and vascular systems require a complex network to properly perform their functions. The processes involved in creating this network rely on coordinated pathways, often activated through common protein/receptor systems, which lead to cytoskeletal remodelling. In general, neuronal and vascular cells respond to extracellular stimuli in the form of soluble secreted proteins, which interact with surface receptors to mediate attraction or repulsion towards the source of the secreted proteins. This process, called guidance, is regulated by seven families of receptors and their respective ligands, which influence each other and can act on the neuronal system, the vascular system or both.Structural information about the extracellular region of many of these receptors, and how signal is relayed across the membrane, is lacking.This study is focused around the extracellular domain of two single-pass transmembrane receptors of the Roundabout and UNC5 protein families that are majorly involved in angiogenesis: Robo4 and UNC5B.Based on the findings of this study, the Robo4 and UNC5B extracellular domains are extensively glycosylated with N-linked glycans of the complex type. Site-directed mutagenesis of the predicted Robo4 glycosylation sites disrupts protein expression, indicating that they are necessary for protein stability and passage through the glycosylation pathway might be necessary for correct folding. MALS and SAXS data show that in solution the Robo4 extracellular domain is a flexible monomer with extended shape. Several Fabs binding to the extracellular domain of Robo4 were characterised, with the expectation to identify those Fabs that could inhibit the reported Robo4/UNC5B interaction for further characterisation. Complex formation was verified by SEC-MALS and SAXS, and interaction constants were determined using SPR. Crystals of some Robo4 extracellular domain/Fab complexes were produced, although the structure of the complex could not be solved at the present time.Despite a study by another group showing otherwise, pull-down, SEC-MALS and SPR experiments show that the Robo4 and UNC5B extracellular domains do not interact with each other. It is proposed that the difference may be caused by different glycosylation patterns specific to the cell lines used for each study, or by an undetected third party necessary for interaction. This, however, still requires further study. SEC-MALS analysis showed that the UNC5B extracellular domain is a monomer in solution and its crystal structure was solved at 3.4 Å resolution. Comparison to the existing structures of human UNC5A and rat UNC5D shows striking similarities and a high degree of evolutionary conservation of the Ig domains might be indication of the importance of this region, which is responsible for binding to the guidance cue Netrin. Although the Netrin binding region is known to be within the Ig domains, the precise binding site has not yet been determined, but it might be located in proximity, or within, the negatively charged surfaces present on the Ig domains which are observed in the UNC5B structure.It is hoped that the work presented here will give the basis for better biochemical and structural characterisation of these two receptors.
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Francesco Bisiak. Structural studies of the Roundabout protein family. Biomolecules [q-bio.BM]. Université Grenoble Alpes, 2018. English. ⟨NNT : 2018GREAV006⟩. ⟨tel-01807970⟩

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