Use this URL to cite or link to this record in EThOS:
Title: Tool development to study ubiquitination machinery
Author: Ward, Jennifer
ISNI:       0000 0004 5994 2828
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
Availability of Full Text:
Access from EThOS:
Access from Institution:
Ubiquitination is a diverse post-translational modification, involved in a plethora of eukaryotic processes. At least three different enzymes are required for ubiquitination to occur: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin ligase (E3). Conversely, deubiquitinating enzymes (DUBs) regulate the removal of ubiquitin modifications. Together, this enzymatic machinery facilitates a wide array of ubiquitin modifications and further ubiquitin-like modifications. Such modifications play a significant role in the regulation of vital biological processes including proteasomal degradation, DNA damage response (DDR) and NF-κB signalling. Ubiquitination machinery can be studied using a chemical proteomics approach. Activity-based probes (ABPs) which covalently trap enzymes can be employed. Such probes possess bioorthogonal handles, which on ligation to complementary reporter groups enable enrichment of probe-enzyme complexes prior to LC-MS/MS, western blot, or fluorescent analysis. The design, synthesis, and development of six inhibitor-inspired ABPs for ubiquitin machinery is described. Both small molecule and peptide probes were explored, and assessed for their ability to act as ubiquitin machinery probes both in intact cells and cell lysate. Spike-in SILAC methodology was employed to quantify probe targets under competitive conditions against parent inhibitors, and in a DNA damage response model. An alternative to using literature inhibitors is to derive novel starting points for tool design through a fragment-based drug discovery approach. A high throughput screen against the minimal catalytic core of HOIP, an E3 ligase, is described together with subsequent validation and characterisation of hit fragments by waterLOGSY NMR and Micro-scale thermophoresis, and crystallography attempts. Finally, a model system for the structural analysis of transient enzyme complexes is explored, to further our understanding of these interactions and ultimately to assess the applicability of these complexes for future drug discovery. The design and synthesis of a maleimide trap is described, along with its application to covalently trap an Ub-E2-E3 complex.
Supervisor: Tate, Ed ; Lam, Eric ; Fuchter, Matthew Sponsor: AstraZeneca ; Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral