Structural and biochemical insights into members of the kinesin and ubiquitin ligase families

Klejnot, Marta (2013) Structural and biochemical insights into members of the kinesin and ubiquitin ligase families. PhD thesis, University of Glasgow.

Abstract

The thesis focuses on characterisation of two different protein families, kinesins and ubiquitin ligases, that are involved in different biological processes. Kinesins constitute a superfamily of microtubule-based motor proteins, fulfilling important cellular functions, ranging from intracellular transport to cell division. They also play a role in primary cilia and Hedgehog signalling. Ubiquitin ligases are enzymes that catalyse the transfer of ubiquitin to the lysine residues of the substrate. Marking protein substrates with ubiquitin alters their functions and fates. With nearly 700 different ubiquitin ligases in humans, they control a vast array of cellular processes. The first part of this thesis summarises results relating to kinesins. Protocols for creation of a panel of kinesin motor domains, a useful tool for anti-mitotic inhibitors specificity testing are presented. Furthermore, biochemical, kinetic and structural features of a poorly described kinesin motor protein - Kif15 are reported. Kif15's motor domain structure is described and compared to Eg5's catalytic core. Moreover, the influence of Eg5 inhibitors on Kif15 ATPase activity is investigated. Scouting for small molecules targeting Kif15 is also performed. Kif15 interaction with microtubules in various nucleotide-bound states is characterised. The possibility of a secondary microtubule binding site in the tail of Kif15 is examined. The binding of Kif15 with partner proteins is also investigated. Additionally the high-resolution structure of the human Kif7 motor domain is presented, providing the first step towards structural characterisation of this Kinesin-4 family member. The second part of this thesis concerns a ubiquitin ligase, Trim28. Trim28 was first reported as a transcription corepressor, working by recruiting proteins that drive the heterochromatin state, whilst its mechanism of action as a ubiquitin ligase remains elusive. Attempts to crystallise and determine the three-dimensional structure of Trim28 are described. Additionally, Trim28 functioning as an E3 ubiquitin ligase, and its interactions with E2s, KRAB domains of various zinc finger proteins and with the Mage-C2 protein are investigated. The results provide the foundation for future studies on Trim28.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: kinesin, Kif15, Kif7, ubiquitin ligase, Trim28
• Subjects: Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Huang, Dr. Danny
• Date of Award: 2013
• Depositing User: Marta Klejnot
• Unique ID: glathesis:2013-5108
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Jun 2014 10:39
• Last Modified: 04 Jun 2014 10:59
• URI: https://theses.gla.ac.uk/id/eprint/5108