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Structure‐Activity Relationship of Peptide Inhibitors of the Wnt/frizzled interaction
Author(s) -
Blankesteijn W. Matthijs,
Hermans Kevin CM,
FernandezLlamazares Ana Iris,
Kriek Nicole,
Timmerman Peter
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.931.13
Subject(s) - cysteine , wnt signaling pathway , frizzled , peptide , xenopus , chemistry , wnt3a , amino acid , index finger , peptide sequence , biological activity , biochemistry , biology , in vitro , anatomy , signal transduction , gene , enzyme
Crystallization of the Xenopus Wnt8 in complex with the cysteine rich domain (CRD) of Frizzled8 has shown that the interaction of Wnt and frizzled resembles the outline of a hand with a thumb and index finger grasping the CRD at two opposing sites. Previous experiments from our group have shown that both the thumb and the index finger fragments can interfere with the Wnt/frizzled interaction at micromolar concentrations. In this follow‐up study, we investigated the structure‐activity relationships of these fragments by changing their size and composition. Moreover, we studied the contribution of the disulfide bonds that are formed between the cysteine residues present in both fragments. Methods Peptides resembling the thumb and index finger of Wnt3a and Wnt5a were synthesized and tested in HEK293 and 3T3 cells expressing the TOPFlash reporter. Canonical Wnt signaling was induced by addition of Wnt3a conditioned medium. Results A gradual decrease of the length of the thumb‐derived sequence from 22 to 13 amino acids resulted in a 40% loss of antagonistic effect at 1.10 −5 M. Palmitoylation of the thumb was found to be essential for biological function. For the index finger‐derived peptide, antagonistic properties remained constant during gradual reduction from 23 to 13 amino acids, but a further reduction resulted in complete loss of antagonistic activity. For both fragments the disulfide bonds most proximal to the tip were important for antagonistic activity whereas the distal disulfide bonds were dispensable. Scrambled peptides did not have antagonistic properties. Conclusion The antagonistic properties of peptide fragments of the thumb and index finger of Wnt peptides depend on the length and proper tertiary structure. Support or Funding Information Supported by the Dutch Heart Foundation (2010B196) and Cyttron (FES0908)