Discovery and characterization of a novel Deubiquitinase ZUP1

Abstract

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

Date of Award	2021
Original language	English
Awarding Institution	University of Dundee
Supervisor	Yogesh Kulathu (Supervisor)