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An investigation into the role of LUBAC and linear ubiquitin chains in antibody dependent intracellular neutralisation


Type

Thesis

Change log

Authors

Green, Christopher 

Abstract

Antibody dependent intracellular neutralisation (ADIN) mediates the rapid proteasomal degradation of antibody-bound viruses. It requires TRIM21, an Fc receptor and E3 ubiquitin ligase that resides in the cytosol. Binding of TRIM21 to the exposed Fc portion of an antibody that is bound to an internalised virus results in ubiquitin signalling and proteasomal degradation of the virus. Viruses are large, complex structures. To facilitate their degradation, it has been established that VCP, an unfoldase, is recruited. VCP provides the capability to degrade these large viruses. I sought to understand how VCP is recruited to the ADIN pathway.

I performed an siRNA screen targeting known VCP adaptors using a TRIM21- dependent adenovirus neutralisation model. I found that HOIP, the only E3 ubiquitin ligase capable of generating linear ubiquitin chains, is required for efficient neutralisation of adenovirus by TRIM21. Using a small molecule inhibitor of HOIP, I found that its E3 ligase ability is required for its function in ADIN. I also tested various domains of HOIP to identify which are required for ADIN. I found that the ability of HOIP to bind ubiquitin chains and its ability to bind VCP are both essential to its role in ADIN. I tested the role of HOIP and linear ubiquitination in other ADIN models of TRIM21-mediated degradation. I found that HOIP and linear ubiquitination are not required for degradation of smaller proteins. This is consistent with previous studies that show that VCP is not recruited to TRIM21 when smaller proteins are targeted for degradation. I also attempted to show that HOIP was required during antibody- mediated protection against tau seeding. This is a novel use of ADIN and shows promise in immunotherapy against Alzheimer’s disease. I found that siRNA technology was not suitable for this assay.

Overall, I have demonstrated that HOIP and linear ubiquitin chains promote recruitment of VCP to ubiquitinated TRIM21. This promotes the efficient degradation of the antibody-bound target. However, I have also found that this is substrate- dependent, favouring complex substrates such as viruses.

Description

Date

2023-04-16

Advisors

McEwan, William
Duce, James

Keywords

Adenovirus, Antibody, HOIP, Linear ubiquitin, LUBAC, M1 ubiquitin, OTULIN, Tau, Tau seeding, TRIM21, Ubiquitin, VCP, Virology

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Alzheimer's Research UK (ARUK-PhD2018-051)
Alzheimer's Research UK PhD Scholarship: ARUK-PhD2018-051