Discovery and characterization of a novel Deubiquitinase ZUP1

Deubiquitinases (DUBs) regulate the post-translational protein modification<br/>ubiquitylation, which is involved in various cellular processes including protein<br/>degradation, an array of signalling pathways and the DNA damage response.<br/>The recent discovery of a fifth family of cysteine protease DUBs highlighted the<br/>possibility that new components of the ubiquitin system remain to be<br/>discovered. Hence, I used Activity-Based Protein Profiling to characterise the<br/>expression and activity of ubiquitin and ubiquitin-like (UBL) proteases.<br/>Excitingly, I identified a potentially novel, hitherto uncharacterised protease<br/>ZUFSP (Zinc finger with UFM1-specific peptidase domain protein) that reacts<br/>only with a ubiquitin probe, and not with other UBL probes. My data show that<br/>the identified protein is indeed an active DUB that cannot be classified as<br/>belonging to any of the existing families. As a consequence of my findings, the<br/>protein was renamed as ZUP1 (Zinc finger Ubiquitin Protease 1).<br/>Further work presented in this Thesis focussed on elucidating the biochemical<br/>properties and cellular function of this novel DUB. Thus, we solved the structure<br/>and performed biochemical characterisation of ZUP1, discovering a novel<br/>ubiquitin binding domain which is essential for its DUB activity and linkage<br/>preference. I showed that ZUP1 specifically cleaves K63-linked polyUb chains,<br/>but also binds different linkage types including K48 polyUb. I investigated<br/>whether this feature could be linked to heterotypic polyUb recognition. Indeed,<br/>I demonstrated that ZUP1 hydrolyses both linkages within a K48-K63 branched<br/>chain. I performed proteomic analyses which, together with collaborative work,<br/>revealed a potential function for ZUP1 in maintaining genome integrity and the<br/>DNA damage response. My preliminary data shows that ZUP1 can be<br/>ubiquitylated and degraded in a proteasome-depended manner upon DNA<br/>damage induction. Utilising CRISPR/Cas9 technology, I generated ZUP1 KO<br/>cell lines which were used for proteomic experiments to identify potential<br/>substrates of ZUP1. Overall, I generated biochemical data that gives an insight<br/>into the mechanism of polyUb recognition and catalysis by ZUP1. Moreover, the<br/>results presented in this work can help to elucidate the cellular function and<br/>regulatory mechanisms of this newly discovered DUB.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2021

Author dc:creator

• Kwasna, Dominika Beata

Advisor dc:contributor.advisor

• Kulathu, Yogesh

• Deubiquitynase
• Ubiquitin
• ZUP1

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/ab49bfb6-eabb-44f2-8833-fd4c0f92a5f7

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/ab49bfb6-eabb-44f2-8833-fd4c0f92a5f7