Structural and biochemical characterisation of p14ARF – E3 ubiquitin ligase complexes

Kowalczyk, Dominika (2019) Structural and biochemical characterisation of p14ARF – E3 ubiquitin ligase complexes. PhD thesis, University of Glasgow.

Abstract

Post-translational modifications are a common mechanism in defining proteins role, fate and engagement in different intracellular processes. One of such modifications is ubiquitination – attachment of a small ubiquitin polypeptide to the target’s lysine residue. This process involves a complex enzymatic cascade, consisting of three enzymes – E1, E2 and E3. As a result, ubiquitin is activated, conjugated and ligated to the target in a well-controlled but poorly understood manner. Ubiquitination can designate proteins for proteasomal degradation, endocytosis or alter their interaction with other partners. Any disorganisation and misregulation of that process can lead to severe disruption of cellular processes, resulting in the development of serious diseases, such as cancer or neurodegenerative disorders.
MDM2 is a RING-E3 ubiquitin ligase, mainly known for its ability to ubiquitinate tumour suppressor p53 and is overexpressed in different types of cancer. MDM2 can act either as a homodimer or a heterodimer, when it’s bound with its homolog – MDM4. Interestingly, despite the high sequence homology, the two proteins behave differently, depending on the dimerisation state. HUWE1 is a HECT-E3 ubiquitin ligase, which has been shown to influence the activity of a range of pro- and anti-apoptotic proteins, such as p53, MCL1 and c-MYC. Both MDM2 and HUWE1 have been reported to be inhibited by p14ARF protein, which has a prominent effect on cell survival and homeostasis.
This thesis presents my work on deciphering how p14ARF influences the activity of MDM2 and HUWE1. I have focused my work on recombinant proteins grown in bacterial expression system, which were further analysed with an implementation of a range of biochemical and structural techniques. Defining an exact mechanism of p14ARF-driven inhibition of MDM2 and HUWE1 could provide long awaited knowledge, relevant for the design of new anti-cancer therapeutics.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Huang, Professor Danny
• Date of Award: 2019
• Depositing User: Dominika Kowalczyk
• Unique ID: glathesis:2019-41040
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Feb 2019 09:56
• Last Modified: 16 Feb 2024 10:15
• Thesis DOI: 10.5525/gla.thesis.41040
• URI: https://theses.gla.ac.uk/id/eprint/41040