Determining the molecular basis for interactions between WW domains of WWP2 and the target regions OCT4 and Smad7

De Bourcier, Danielle (2018) Determining the molecular basis for interactions between WW domains of WWP2 and the target regions OCT4 and Smad7. Doctoral thesis, University of East Anglia.

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WWP2, a NEDD4 E3 ubiquitin ligase, contains four WW domains which display a preference for binding protein substrates with proline-rich motifs. Through these WW domains, WWP2 interacts with different substrates such as PTEN (a tumour suppressor gene often lost in cancers), Smads (involved in the TGFβ signalling pathway, often deregulated in various cancers), and the stem-cell specific transcription factor OCT4 (involved in cancer cell proliferations, dedifferentiation, and chemotherapy resistance). Ubiquitination of the TGFβ receptors is mediated by WWP2-Smad7 complexes, marking them for degradation in the proteasome. WWP2-OCT4 complexes result in degradation of OCT4 in the proteasome. Downregulation of OCT4 occurs during differentiation; however, some malignant cells regain the ability to express OCT4. Overexpression of WWP2 in animal models causes increased tumour spread, and human cancer tissue samples present altered isoform expression patterns for WWP2.

This thesis investigates WW domain structural characteristics and the interactions between these domains and two of their targets, Smad7 and OCT4. Using Nuclear Magnetic Resonance (NMR) spectroscopy, we show that the second WW domain (WW2 - expressed as a fusion protein with solubility tag GB1) is in an intermediate exchange regime when isolated, indicating that it is not fully folded. By observing ligand interaction using NMR, peptides derived from Smad7 and OCT4 are found to bind to the WW2 domain and stabilise its structure. The two peptides have a similar, but not identical, effect on the structure and reveal the respective binding sites of WW2. In addition, NMR spectroscopy has provided structural information of the isolated GB1-WW3 domain. Isothermal Titration Calorimetry (ITC) confirmed interactions between WW2 and its targets and indicates that Smad7 binds with a higher affinity than OCT4.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Users 9280 not found.
Date Deposited: 10 Jun 2019 13:55
Last Modified: 12 Oct 2021 00:38


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