Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.630232
Title: Biochemical and biophysical studies of MDM2-ligand interactions
Author: Wang, Shao-Fang
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2012
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Please try the link below.
Access through Institution:
Abstract:
MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding.
Supervisor: Walkinshaw, Malcolm; Ball, Kathryn Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.630232  DOI: Not available
Keywords: MDM2 ; virtual screening ; ligand ; E3 ligase ; ubiquitination
Share: