Architecture and hydration of the arginine-binding site of neuropilin-1.

Neuropilin-1 (NRP1) is a transmembrane co-receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C-terminal arginine of VEGF and residues in the NRP1-binding site including Tyr297, Tyr353, Asp320, Ser346 and Thr349. We obtained several complexes of the synthetic ligands and the NRP1-b1 domain and used X-ray crystallography and computational methods to analyse atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the six new high-resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand-binding site hydration map of NRP1 was in agreement with the experimentally derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein-ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands' C-terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design and validates the utility of the computational hydration map prediction method in the context of neuropilin. DATABASE: The structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.

Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis.

Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (ß-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning ß-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. ß-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated ß-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that ß-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible ß-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, ß-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. ß-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, ß-AR agonists could be promising anti-angiogenic modulators in skin.

ß2AR antagonists and ß2AR gene deletion both promote skin wound repair processes.

Skin wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and biological processes to regenerate damaged tissue. Previous work has demonstrated that a functional ß-adrenergic receptor autocrine/paracrine network exists in skin, but the role of ß2-adrenergic receptor (ß2AR) in wound healing is unknown. A range of in vitro (single-cell migration, immunoblotting, ELISA, enzyme immunoassay), ex vivo (rat aortic ring assay), and in vivo (chick chorioallantoic membrane assay, zebrafish, murine wild-type, and ß2AR knockout excisional skin wound models) models were used to demonstrate that blockade or loss of ß2AR gene deletion promoted wound repair, a finding that is, to our knowledge, previously unreported. Compared with vehicle-only controls, ß2AR antagonism increased angiogenesis, dermal fibroblast function, and re-epithelialization, but had no effect on wound inflammation in vivo. Skin wounds in ß2AR knockout mice contracted and re-epithelialized faster in the first few days of wound repair in vivo. ß2AR antagonism enhanced cell motility through distinct intracellular signalling mechanisms and increased vascular endothelial growth factor secretion from keratinocytes. ß2AR antagonism promoted wound repair processes in the early stages of wound repair, revealing a possible new avenue for therapeutic intervention.