Novel Approaches to Probe the Activity of Deubiquitinating Enzymes
Citation:
Taylor, Neil, Novel Approaches to Probe the Activity of Deubiquitinating Enzymes, Trinity College Dublin.School of Chemistry, 2021Download Item:
Abstract:
Ubiquitination is a highly conserved post-translational modification that regulates a
multitude of critical cellular events. This process is orchestrated by a complex
enzymatic network. Deubiquitinating enzymes (DUBs) are responsible for removal
of ubiquitin from its conjugates. There are over 100 DUBs expressed in eukaryotes
and several diseases are associated with their dysregulation, including cancer and
neurodegeneration. Activity-based probes (ABPs) have been developed as an
effective strategy to study DUBs.
ABPs targeting these enzymes are typically based on a ubiquitin scaffold and
incorporate an electrophilic group that reacts with an active site cysteine. These
probes have greatly enhanced the mechanistic and structural understanding of
DUBs but offer no external control over the timing of the reaction. In this work, ABPs
consisting of a monoubiquitin recognition element and warheads with reactivity
previously unexplored in the context of ABPs were designed and synthesised. The
reactivity of these novel ABPs with DUBs was examined and new labelling
strategies were developed to improve the study of these enzymes.
New electrophilic probes with fluoride reactive groups were designed and
synthesised. These probes were tested alongside previously reported electrophilic
probes and demonstrated negligible reactivity with DUBs.
Latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated
thiol-ene coupling mechanism were developed. A novel labelling methodology was
developed for these alkene-functionalised probes and reactivity was demonstrated
against recombinant DUBs and endogenous DUBs within cell lysate. Specificity of
this probe labelling was confirmed using inhibitor and proteomic studies. Novel
assays to probe reversible and irreversible inhibitors for this enzyme were also
demonstrated.
In order to enhance the biocompatibility of this methodology, a milder source of UV
light was used to initiate the reaction between DUBs and the alkene-functionalised
probes. Alternative radical initiators were examined for a visible light-mediated
thiol-ene reaction. Successful labelling of recombinant DUBs was achieved in both
strategies, but visible light activation was limited by off-target reactivity in more
complex systems. The visible light-mediated thiol-ene reaction and the thiol-yne
reaction were also explored for non-templated protein conjugation, affording modest
coupling in both cases.
Overall, the novel electrophilic probes presented do not improve upon existing
probes of similar reactivity. However, the work on alkene functionalised probes
enables more finely resolved investigations of DUB activity in complex systems. In
contrast to existing cysteine reactive probes, control over the timing of the
enzyme-probe reaction is possible for the alkene functionalised probes as they are
completely inert under ambient conditions, even upon probe binding. This is
expected to help provide a better understanding of these enzymes and aid in the
study and development of novel inhibitors. The visible light-mediated thiol-ene and
thiol-yne reactions were found to have limited applications for bioconjugations.
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Trinity College Dublin
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https://tcdlocalportal.tcd.ie/pls/EnterApex/f?p=800:71:0::::P71_USERNAME:TAYLORNEDescription:
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Author: Taylor, Neil
Advisor:
McGouran, JoannaPublisher:
Trinity College Dublin. School of Chemistry. Discipline of ChemistryType of material:
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