Structure-function analysis of the mouse Delta1 gene in vivo and in vitro
Final Report Abstract
Notch signal transduction is an evolutionary conserved essential signaling pathway that mediates cellto-cell communication between adjacent cells. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. The N-terminal domain, together with a so-called DSL domain, and the adjacent three EGF repeats of ligands are necessary and sufficient for binding to and full activation of Notch in various assays in vitro. Notch ligands have additional EGF repeats in their extracellular domain whose significance for Notch activation is unclear. EGF repeats contain three characteristic disulfide bridges that stabilize each individual domain, which is considered as an independently folding protein module. Therefore, disruption of disulfide bonds in individual EGF repeats should be a means to disrupt the structural integrity of individual modules without affecting the intrinsic structure of neighboring repeats. This should allow one to study the relevance of the structural integrity of individual EGF repeats in the context of full length membrane-bound DLL1 for Notch activation. To shed light on the relevance of all endogenous EGF domains in the murine Notch ligand DLL1 for Notch activation, we have computed the structural and dynamic changes of individual EGF repeats, and analyzed the consequences of the predicted structural alterations for DLL1-mediated Notch signaling both in cultured cells and under physiological conditions in vivo. We have individually disrupted the structural integrity of each of the eight EGF repeats that are present in DLL1 and systematically evaluated the consequences of mutations in individual EGF repeats in vitro by Notch activation assays and in vivo by generation and phenotypic characterization of an allelic series of Dll1 in mice. Our Molecular Dynamics Simulations showed that the wild type DLL1 protein is more flexible than thought, and that the mutation of two cysteine residues causing elimination of two intra-domain disulfide bridges of any EGF repeat disrupts their intrinsic secondary structure to varying degrees which suggests that the mutations impact on the conformation of the DLL1 protein. In in vitro assays the mutation of each individual EGF repeat attenuated DLL1 activity, the reduction correlating mostly with the anticipated severity of the disrupted secondary structure of a particular EGF repeat. The activation of the NOTCH1 and NOTCH2 receptors were similarly affected in vitro. Our in vivo analyses showed that early developmental processes depending on DLL1-mediated NOTCH activation are differently affected by these mutations: somitogenesis was most sensitive to EGF mutations of DLL1 showing a range of phenotypes. Myogenesis, neurogenesis, and establishment of left right asymmetry were less sensitive and responded similarly to reduced DLL1 function apparently in all or none fashion. This analysis demonstrates that also the structural integrity of each individual EGF repeat of DLL1 outside the Notch interaction domain is necessary for full ligand activity, and early developmental processes in mouse embryos differ in their sensitivity towards structural changes of DLL1.
Publications
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(2009) DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113: 5680–5688
Sörensen I, Adams RH, Gossler A
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(2014) S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development. Molecular and Cellular Biology 34: 1221–1233
Braune E-B, Schuster-Gossler K, Lyszkiewicz M, Serth K, Preusse K, et al.
