Hess, M; (2016) Biophysical and biochemical investigations of CARD-CARD interactions. Doctoral thesis , UCL (University College London).

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Abstract

Innate and adaptive immunity are regulated by complex signalling pathways that require the assembly and activation of apoptotic and inflammatory signalling complexes, which ultimately induce gene expression and alter cellular behaviour. Key players in regulating the assembly of many of these signalling complexes are CARD containing proteins that are members of the death domain superfamily. However, the molecular mechanisms that underline the function and regulation of CARDs remain unclear. This project was aimed to investigate the biochemical and biophysical characteristics of CARD-CARD interactions to gain insights into the binding specificity and/or stoichiometry of CARD-mediated protein complexes and specifically, to elucidate if there is a common mode of interaction within this protein subfamily. It was known that CARDs have a tendency to aggregate when purified individually, therefore all human CARDs were prepared for small scale expression and solubility screening with the aim to perform co-expression and pull-down studies. These screens identified the highly soluble, yet structurally and biophysically uncharacterised CARMA1 CARD. Consequently, one focus was set on the signalling complexes involving CARMA1 and CARD9, both of which bind BCL10 via CARD-CARD interactions. Preliminary work had been performed in the group focusing on NOD2 and RIP2, therefore, these two proteins were taken as a second system for further investigations with the aim to describe the intramolecular interaction between the tandem CARDs of NOD2 and its intermolecular interaction with the CARD of RIP2 in molecular detail. Further insights into the binding surfaces involved in these interactions were gained using mutagenesis combined with biophysical assays. To follow up on the newest development in the field concerning CARDs forming filamentous assemblies, this work tested both, RIP2- and BCL10-CARD for their ability to polymerise using fluorescence polymerisation assays. However, no polymerisation could be detected in this work, questioning the relevance of filament formation as a general behaviour of CARDs.

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