F‐domain valency determines outcome of signaling through the angiopoietin pathway

Abstract

Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F‐domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor‐binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell–cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F‐domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F‐domains: Scaffolds presenting 3 or 4 F‐domains have Ang2‐like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F‐domains have Ang1‐like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F‐domains display super‐agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super‐agonist nanoparticles reduced blood extravasation and improved blood–brain barrier integrity four days after a controlled cortical impact injury.

Top-down design of protein architectures with reinforcement learning

“…To assess the efficacy of the designed capsids in activating cellular signaling pathways by clustering cell surface receptors, we fused 60 copies of the angiopoietin 1 (Ang1) F domain (Fd), which binds the Tie2 receptor, to RC_I_1-H11 using SpyTag-SpyCatcher conjugation ( 14 , 18 , 43 ) (see the materials and methods and fig. S27).…”

“…S28, A to C) and stabilizing nascent blood vessels formed from human umbilical vein endothelial cells (HUVECs; Fig. 5E) ( 43 – 49 ). The Fd-displaying capsids (0.16 nM RC_I_1-H11-Fd) elicited stronger responses than a 10-fold greater concentration of a much larger F-domain–presenting icosahedral nanoparticle (I53-50) ( 12 , 43 ); the elevated potency likely results from the higher surface display density [to facilitate comparison, concentrations at the bottom of Fig.…”

“…5E) ( 43 – 49 ). The Fd-displaying capsids (0.16 nM RC_I_1-H11-Fd) elicited stronger responses than a 10-fold greater concentration of a much larger F-domain–presenting icosahedral nanoparticle (I53-50) ( 12 , 43 ); the elevated potency likely results from the higher surface display density [to facilitate comparison, concentrations at the bottom of Fig. 5D are in terms of Fd monomer (0.16 nM capsid × 60 Fd copies per capsid = 10 nM Fd)].…”

Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery

“…Targeted stimulation or inhibition of cell signaling receptors is the key mechanism of many therapeutics ranging from receptor tyrosine kinase inhibitors to bispecific antibodies. Clinical translation of many such therapeutics is hindered by challenges with ligand stability, pharmacokinetics, biodistribution, and hepatotoxicity. − Displaying receptor agonists or antagonists on protein nanoparticles offers several opportunities to address these challenges: the (ant)­agonist can be stabilized by scaffolding on a solid foundation, the signaling magnitude can be tuned through altering display valency and nanoparticle architecture, and the biodistribution profile can be improved through optimizing the physicochemical features of the nanoparticle. ,,− Inspired by receptor signaling applications based on VLPs, ferritin and computationally designed nanoparticles have recently been engineered to display a variety of therapeutic receptor signaling domains. Examples include interleukin-4 receptor (IL-4R)-targeting peptides to ameliorate asthma symptoms, antimesenchymal epithelial transition (MET) peptide pharmacophores to stimulate hepatocyte growth factor receptor, death receptor ligands (TRAIL) to kill tumor cells, and angiopoietin-agonizing antibodies to promote angiogenesis (Figure ).…”

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

“…Clustering of cell surface receptors can enhance and sustain activation in response to an extracellular signal, and there is considerable interest in technologies to manipulate receptor clustering. [1][2][3][4][5] Designed protein assemblies have been used to drive receptor clustering; [6][7][8] particularly useful are oligomers made from idealized repeat proteins because their length can be systematically varied by addition of repeat units. 9 Previous work has largely focused on designs that bring together two receptor subunits, [10][11][12][13] but higher order receptor assemblies are thought to function in a number of signaling systems.…”