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Stapled helix8/6/2023 (G) HEK293T cells transfected with Topflash-luciferase were treated with Wnt3a CM and SAHPAs at the indicated concentrations for 12 h and then harvested for luciferase measurement. (F) HEK293T cells were treated with Wnt3a conditioned medium (CM) and SAHPAs (40 μM) for 12 h and then harvested for immunoblotting. (E) Coimmunoprecipitation of endogenous β-catenin with Axin2 in the presence of SAHPAs (40 μM). The precipitants were immunoblotted with anti-GST antibody, and GST-β-catenin (133-665) inputs were shown by Coomassie staining. (D) Biotinylated-peptides-bound streptavidin beads were employed to pull down bacterially-expressed recombinant GST-β-catenin (133-665) fragment. (C) Sequences of Axin (469-482)-derived SAHPAs. Two non-natural alkenyl amino acids (R8 and S5) are incorporated at two positions in the peptide chain and then cross-linked by ring-closing olefin metathesis. (A) The crystal structure of the β-catenin-Axin complex with highlights of Axin (469-482) (green) and the shallow groove of β-catenin (purple and blue). Circular dichroism analyses revealed that while Axin (469-482) displays 28% α-helical content, SAHPA1 and SAHPA2 have 34% and 57% α-helicity, respectively ( Supplementary information, Figure S1).ĭesign of stapled α-helical peptides to activate Wnt/β-catenin signaling. Two stapled α-helical peptides targeting the Axin-β-catenin complex (SAHPA), SAHPA1 and SAHPA2, were generated ( Figure 1C). We then synthesized the stapled peptides by incorporation of non-natural amino acids at neighboring positions along one face of the α-helix, followed by ring-closing olefin metathesis ( Figure 1B). The crystal structure of Axin reveals that its β-catenin-binding domain (Axin (469-482)) forms a continuous α-helix that fits into a shallow groove of β-catenin formed by the third helices of β-catenin armadillo repeats 3 and 4 4 ( Figure 1A), suggesting that the Axin-β-catenin interaction is suitable for targeting by hydrocarbon-stapled α-helical peptide mimetics. To design cell-permeable stapled peptides that can activate Wnt/β-catenin signaling with good selectivity, we aim to disrupt the Axin-β-catenin interaction. In the complex, Axin directly interacts with and targets β-catenin for GSK3β-mediated phosphorylation and subsequent ubiquitination and degradation 3. In the canonical Wnt/β-catenin signaling pathway, β-catenin stability is regulated by the destruction complex containing Axin, adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β). Compared with small molecules, stapled peptides have larger surface areas, and therefore can selectively disrupt protein-protein interactions and have been successfully employed to modulate NOTCH and p53 signaling 2. In vivo, stapled peptides can penetrate through cell membranes to attack the intracellular targets and display high protease resistance. This method has been demonstrated to efficiently increase the helical propensity and the binding ability of peptides. 2 to mimic the α-helix conformation of folded proteins. The design of hydrocarbon-stapled peptides was developed by Verdine et al. Additionally, as the Wnt/β-catenin signaling pathway is critical for the maintenance of embryonic stem cells and multiple types of adult stem cells, agonists or antagonists of Wnt/β-catenin signaling may provide useful tools for the studies of stem cell self-renewal and differentiation, and tissue regeneration. The design of organic compounds that modulate Wnt/β-catenin signaling constitutes an interesting strategy for therapeutic intervention of this key pathway. Aberrant activation of the Wnt/β-catenin signaling pathway has been implicated in the development of a broad spectrum of tumors, while attenuation of this pathway contributes to a number of human diseases including osteoporosis, neurodegenerative diseases, diabetes and Joubert syndrome 1. This pathway plays important roles in embryogenesis, adult tissue homeostasis and tissue regeneration. The canonical Wnt/β-catenin signaling pathway orchestrates cell morphology, motility, proliferation and differentiation.
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