Репозиторий Университета

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein-protein interactionWWdomains. The WW4 domain-containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial-mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-DHECT) comprising WW3-WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis.

© 2018 Elsevier Ltd  Five lactam chemotypes amenable by the Castagnoli-Cushman reaction of imines and cyclic anhydrides have been investigated for their ability to activate p53 tumor suppressing transcription factor thus induce apoptosis in p53 + cancer cells. A virtual library of 1.07 million chemically diverse compounds based on these scaffolds was subjected to in silico screening first. The compounds displaying high docking score were visually prioritized to identify the best-fitting compounds, i. e. the ones which adequately mimic the interactions of clinical candidate inhibitor Nutlin-3a. These 38 compounds were synthesized and tested for apoptosis induction in p53 + H116 cancer cells to identify 9 potent apoptosis-inducers (two of them exceeding the activity of Nutlin-3a) which belonged to four different chemotypes. The activation of p53 involved in the proapoptotic activity observed was supported by effective induction of EGFP expression in human osteocarcinoma U2OS-pLV reporter cell line. Moreover, the two most potent apoptosis inducers displayed antiproliferative profile identical to several known advanced p53 activators: they inhibited the growth of p53 +/+ HCT116 cells in much lower concentration range compared to p53 −/− HCT116 cells.