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Title: Substrate recognition by the 26S proteasome
Author: Kisonaite, Migle
ISNI:       0000 0004 7968 626X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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The 26S proteasome is a large protein complex found in all eukaryotes. It controls the degradation of a wide range of proteins in the cell, and thus it is crucial for homeostasis, regulated cell division and apoptosis. It is known that specific signals (called degrons) on the substrates are recognised by the proteasome, including ubiquitin and a disordered region. The main focus of this thesis is to better understand how the ubiquitin containing degron binds to intrinsic ubiquitin receptors in the 26S proteasome and how the architecture of the degradation signal influences the affinity of the interaction. Firstly, I optimised the purification of the human 26S proteasome, resulting in a high yield and purity sample. I showed that my 26S proteasome sample is fully functional - can recognise and bind the substrate, unfold it, translocate it into the proteolytic chamber and digest it to small peptides. I also investigated the stability of the 26S proteasome and discovered some novel insights about divalent cation effects on the proteasome activity. During this project I prepared and optimised a large number of model degrons with variable lengths and composition of their disordered regions. Degron optimisation was an iterative process, thus during my project I designed, cloned, purified and characterised more than 50 different proteins. I found the best binding degron, co-purified it with the human 26S proteasome and obtained a homogeneous sample suitable for structural studies. Finally, I solved two cryo-EM structures of the degron-bound 26S proteasome. The structures revealed two novel conformations of the proteasome and the subunits responsible for the interaction with the ubiquitin. Using the solved molecular models, together with biophysical data obtained during the project, I explored the mechanistic details of the ubiquitin recognition and conformational changes of the 26S proteasome upon the interaction with a degradation signal.
Supervisor: da Fonseca, Paula Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: 26S proteasome ; cryo-electron microscopy ; protein degradation ; degron ; proteolysis