Selectional and mutational scope of peptides sequestering the Jun-Fos coiled-coil domain |
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Authors: | Hagemann Urs B Mason Jody M Müller Kristian M Arndt Katja M |
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Affiliation: | Institute for Biology III, Albert-Ludwigs-University of Freiburg, Schaenzlestrass 1, D-79104 Freiburg, Germany |
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Abstract: | The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability to induce tumorigenesis is well documented. Thus, AP-1 represents an interesting therapeutic target. We selected peptides from phage display and compared their ability to disrupt the cFos/cJun interaction to a previously described in vivo protein-fragment complementation assay (PCA). A cJun-based library was screened to enrich for peptides that disrupt the AP-1 complex by binding to the cFos coiled-coil domain. Interestingly, phage display identified one helix, JunWPh1 [phage-selected winning peptide (clone 1) targeting cFos], which differs in only 2 out of 10 randomized positions to JunW (PCA-selected winning peptide targeting cFos). Phage-selected peptides revealed higher affinity to cFos than wild-type cJun, harboring a Tm of 53 °C compared to 16 °C for cFos/cJun or 44 °C for cFos/JunW. In PCA growth assays in the presence of cJun as competitor, phage-selected JunWPh1 conferred shorter generation times than JunW. Bacterial growth was barely detectable, using JunWPh1 as a competitor for the wild-type cJun/cFos interaction, indicating efficient cFos removal from the dimeric wild-type complex. Importantly, all inhibitory peptides were able to interfere with DNA binding as demonstrated in gel shift assays. The selected sequences have consequently improved our ‘bZIP coiled-coil interaction prediction algorithm’ in distinguishing interacting from noninteracting coiled-coil sequences. Predicting and manipulating protein interaction will accelerate the systems biology field, and generated peptides will be valuable tools for analytical and biomedical applications. |
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Keywords: | AP-1, activator protein-1 bCIPA, bZIP coiled-coil interaction prediction algorithm BSA, bovine serum albumin C, core pairing CD, circular dichroism DHFR, dihydrofolate reductase EDTA, ethylenediaminetetraacetic acid EMSA, electrophoretic mobility shift assay ES, electrostatics GST, glutathione S-transferase HP, helix propensity JunW, PCA-selected winning peptide targeting cFos JunWPhx, phage-selected winning peptide (clone x) targeting cFos PCA, protein-fragment complementation assay PEG, polyethylene glycol PV hypothesis, peptide Velcro hypothesis SEC, size-exclusion chromatography TPA, 12-O-tetradecanoylphorbol-13-acetate TRE, TPA-response element |
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