Investigation into the regulatory mechanisms of the NOD2 signalling pathway
AuthorStafford, Che Alan
The Walter and Eliza Hall Institute of Medical Research
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