Investigation into the regulatory mechanisms of the NOD2 signalling pathway
AuthorStafford, Che Alan
The Walter and Eliza Hall Institute of Medical Research
MetadataShow full item record
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr. Che Alan Stafford
Nucleotide-binding oligomerisation domain-containing (NOD) receptor 2 (NOD2) is a critical intracellular sentinel for Gram-positive and Gram-negative bacteria through the recognition of the bacterial cell wall component muramyl dipeptide (MDP). NOD2 signalling relies on the recruitment and ubiquitylation of the adaptor kinase receptor interacting protein kinase 2 (RIPK2). Multiple E3-ubiquitin ligases have been reported as vital components of the NOD2 signalling complex, including X-linked inhibitor of apoptosis (XIAP), and cellular IAP1 and 2 (cIAP1 & cIAP2). All of these E3 ligases have been shown to have the ability to bind and ubiquitylate RIPK2, yet their physiological role in the NOD2 pathway is unclear. Only a few members of the NOD2 signalling complex have been identified and validated under endogenous conditions, due to a lack of molecular tools. Critically, over-expression of signalling components is an imperfect way to analyse this complex, since it leads to the activation of the pathway independent of MDP engagement. In this thesis, I have identified novel molecular mechanisms of how NOD2 signalling is regulated. Firstly, I clarified that XIAP is the only IAP required for signalling immediately downstream of NOD2, whilst cIAP1 and cIAP2 are dispensable. Through genetic removal of TNFR1 pathway members, I discovered a TNFR1-dependent autocrine amplification loop, following NOD2 activation, which implicates cIAP1 and cIAP2 in the overall cytokine response to MDP. In the second project, I aimed to better understand how the kinase activity of RIPK2 impacts upon NOD2 signalling. For this, I purified and crystallised the recombinant human RIPK2 kinase domain and observed a potential dimerisation interface which may be critical for signalling. This work has established a structural biology approach that can be used for future development of RIPK2 inhibitors. Finally, using a combination of cell biology, biochemistry and chemical biology, I developed and optimised a functionalised MDP-ligand that can endogenously pull-down NOD2 complex members, which will be used to identify novel NOD2 interactors. Together, work from this thesis provides unprecedented clarity surrounding the role of putative E3-ligases in NOD2 signalling, which not only rectified the literature, it will open up many avenues for novel research. This work also establishes the groundwork for structure-led development of future RIPK2 inhibitors, and provides a much-needed endogenous tool to delineate novel interacting partners.
KeywordsNOD2; RIPK2; innate immunity; structural biology; cell biology; bacterial recognition; cell signalling; Muramyl Dipeptide
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- Medical Biology - Theses