Identifying and characterising new targets of inflammatory caspases
File(s)
Author(s)
McCarthy, Callum
Type
Thesis
Abstract
Caspases have pivotal functions in regulating programmed cell death, immunity, and maintaining cellular homeostasis, typically by cleaving their substrates which go on to perform various intra- and extra-cellular functions. Caspase-1 and caspase-4 are major inflammatory caspases that are activated in response to microbial infection and/or danger signals. Even though the mechanisms of caspase-1/4 activation are well-understood, there remains a significant gap in our understanding of the specific cellular proteins that caspase-1/4 target to facilitate inflammasome-mediated processes like pyroptosis, cytokine processing and secretion. In this thesis I set out to phenotypically characterise some of the potential caspase substrates identified in an initial proteomic screen by the Shenoy laboratory. I designed stably silenced and characterised CRISPR/Cas9-derived knockout cell lines as tools to investigate new caspase-targets. My studies focussed on two key proteins, the cytoskeletal protein zyxin, and the ubiquitin-conjugating enzyme UBE2L3. I demonstrate that zyxin can be proteolytically cleaved by caspase-4 in response to LPS-sensing or infection by enteropathogenic E. coli. Substitution of Asp 149 for Ala abolished cleavage, indicating this is the site of proteolysis by caspase-4. I also followed up on previous studies from the group that showed UBE2L3 negatively regulates pro-IL-1β levels in macrophages. I characterised primary macrophages from newly derived Ube2l3-knockout mice and established that loss of Ube2l3 specifically elevated pro-IL-1β protein abundance without affecting caspase-activation or pyroptosis. Taken together, these findings uncovered another layer of complexity in regulating inflammatory responses. Understanding downstream effects of inflammasome activation could lead to the development of targeted therapeutics against inflammasome-driven diseases.
Version
Open Access
Date Issued
2023-09-13
Date Awarded
01/12/2023
Advisor
Shenoy, Avinash
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)