Identification and characterisation of the linear ubiquitin chain assembly complex (LUBAC) as a functionally important component of the CD40 receptor signalling complex

Abstract

CD40 is a member of the Tumour Necrosis Factor (TNF)-Receptor superfamily and plays an important role in maturation and differentiation of dendritic cells and B cells. In B cells, CD40 provides a costimulatory signal and facilitates differentiation, germinal centre formation and isotype switching. So far it has been shown that TNF-Receptor Associated Factors (TRAFs) and cellular Inhibitor of Apoptosis Proteins (cIAPs) are important mediators of CD40 signalling and needed for the activation of Nuclear Factor-κB (NF-κB) and Mitogen- Activated Protein Kinase (MAPK) signalling pathways. However, the exact function of these components and their biochemical interplay are still unknown. Hence, our understanding of the CD40-Receptor Signalling Complex (RSC) which initiates CD40 signal transduction is incomplete. In order to understand the biochemical processes that initiate CD40 signalling a new technique suitable to identify core components of the CD40-RSC was developed in this thesis. This technique is based on the employment of a tandem-tagged recombinant version of CD40 ligand to purify the CD40-RSC in two separate steps. This resulted in higher purity of the receptor complex and allowed for determining the composition of the CD40-RSC by mass spectrometry. This established protocol revealed the recruitment of three novel constituents to the CD40- RSC. HOIL-1, HOIP and SHARPIN are able to form the Linear Ubiquitin Chain Assembly Complex (LUBAC) which catalyses the formation of linear ubiquitin chains. This complex was only recruited to the CD40-RSC when highly ubiquitinated cIAP2 was also present. It was then shown that HOIL-1 and HOIP can bind to ubiquitin chains in vitro and that cIAPs are required for LUBAC recruitment to the CD40-RSC. Specific suppression of LUBAC components by RNA interference or by genetic ablation resulted in an inhibitory effect on CD40 signal transduction, gene induction and isotype switching caused by a defect in NF-κB and MAPK signalling