Mechanisms underlying substrate engagement by TRIM7 and TRIM21 and strategies for re-directing their E3 ligase activity

The work presented in this thesis focuses on two members of the TRIM protein family: TRIM7 and TRIM21. Both proteins are ubiquitin E3 ligases that ubiquitinate and degrade substrates. My work on TRIM7 has uncovered a unique molecular mechanism of substrate selection and explains why TRIM7 is reported to interact with unrelated and diverse substrates, ranging from cellular proteins involved in metabolism to viral proteins. In summary, TRIM7 recognises a dipeptide motif at the C terminus of substrates, so if this sequence is exposed in the folded protein and the substrate is oligomeric, this results in substrate engagement in cells and subsequent degradation. The function of TRIM21 as a cytosolic antibody receptor has been firmly established – it can recognise antibody coated viruses that have managed to enter the cytoplasm and mediate their degradation. This feature of TRIM21 has also been exploited to develop a technology called Trim-Away, where antibodies against endogenous proteins can be introduced to the cytosol of cells, resulting in targeted degradation of these proteins. While it is understood that the RING-E3 domain of TRIM21 is essential for this degradation, along with a number of E2 enzymes, how and why the formation of a TRIM21:antibody:substrate ternary complex results in the degradation of each component is incompletely understood. I have developed and applied *in vitro* assays to monitor ubiquitination of proteins involved in physiological TRIM21 function and in Trim-Away experiments. In both cases, the role of N terminal ubiquitination is explored. Finally, I have developed degrader molecules based on the mechanistic rules underpinning TRIM21 activation. These molecules can be further used as tools for studying endogenous TRIM21 function and potentially developed into therapeutics.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Luptak, Jakub

Advisor dc:contributor.advisor

• James, Leo

• antibody
• drug discovery
• protein degradation
• TRIM
• ubiquitin

Licence

• https://www.repository.cam.ac.uk/bitstreams/aa9b0ef7-6494-4269-a7e1-274efc6ccb08/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0002-9527-8755

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/365599