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The role of tyrosine phosphorylation in inflammasome complex formation and function.

Mambwe, Bezaleel (2018) The role of tyrosine phosphorylation in inflammasome complex formation and function. PhD thesis, University of Sheffield.

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Abstract

The inflammasome is a multi-protein intracellular complex formed following detection of immune ‘danger’ signals (pathogen-associated and damage-associated molecular patterns) that lead to the release of pro-inflammatory cytokines in the presence or absence of pyroptosis. Protein phosphorylation, a reversible post-translational modification of proteins that results in the addition or removal of the phosphoryl group to a specific amino acid residue(s), is important in regulating cellular processes. Tyrosine phosphorylation has been shown to be essential in the regulation of the inflammasome and occurs at the level of the receptors (e.g., NLRP3, AIM2, and NLRC4) and adaptor protein, apoptosis-associated Speck-like protein with a CARD domain (ASC). To identify the role of protein tyrosine phosphatases involved in inflammasome function, the protein tyrosine phosphatase inhibitor, phenylarsine oxide (PAO) was used to assess the activation of NLRP3, AIM2 and NLRC4 inflammasomes. We have found that PAO perturbs the formation of the inflammasome assessed by nucleation of ASC specks, and the processing and release of caspase-1 and IL-1β in both human and murine cells. Furthermore, we have demonstrated that PAO inhibits nigericin-induced global tyrosine dephosphorylation and ASC dephosphorylation implicating tyrosine dephosphorylation of the inflammasome and ASC as a necessary step in inflammasome activation. Furthermore, by utilising site-directed mutagenesis, we identified putative tyrosine residues on ASC required for its function. The mutation of tyrosine residues to a non-phosphorylatable tyrosine mimic, phenylalanine, at residues Y60 and Y137 of ASC results in attenuated IL-1β release but not at Y36. Taken together, we have shown that PAO is a potent inhibitor of ASC dephosphorylation and consequently the NLRP3 and AIM2 inflammasomes suggesting an important role for tyrosine dephosphorylation in the activation of the inflammasome. In addition, we have demonstrated that phosphorylation of ASC at Y60 and Y137 is required for inflammasome function. However, more work is required to identify putative kinases/phosphatases involved in inflammasome regulation.

Item Type: Thesis (PhD)
Keywords: Inflammasome, Phosphorylation, NLRP3, ASC
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.778756
Depositing User: Mr Bezaleel Mambwe
Date Deposited: 10 Jun 2019 08:21
Last Modified: 25 Sep 2019 20:08
URI: http://etheses.whiterose.ac.uk/id/eprint/24113

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