Biochemical and functional characterisation of the Linear Ubiquitin Chain Assembly Complex (LUBAC) as a component of the TNF-Receptor 1 signalling complex

Abstract

Tumour necrosis factor (TNF) plays a critical role in inflammatory processes and is involved in the regulation of immune responses. Depending on the cellular context, TNF initiates a complex cascade of signalling events that can lead to induction of proinflammatory cytokines, cell proliferation, differentiation or cell death. Most of the pleiotropic effects of TNF are mediated by the death-domain (DD) containing TNF-R1. Ligand-induced trimerisation of TNF-R1 leads to the formation of an intracellular multiprotein complex, the TNF-R1 signalling complex (TNF-RSC). To be able to comprehend the complex network of signalling pathways emanating from TNF-R1, the TNF-RSC and its composition need to be understood at the molecular level. Using a modified tandem affinity purification approach, HOIL-1 and HOIP were identified as two novel, functional components of the native TNF-RSC. Together they were shown to form a linear ubiquitin chain assembly complex (LUBAC), catalysing the formation of linear head-to-tail ubiquitin chains. LUBAC mediates ubiquitination of NEMO with linear ubiquitin chains, required for efficient NF-kB activation following TNF stimulation. In this thesis, it could be demonstrated that the stimulation-dependent recruitment of LUBAC to the TNF-RSC is impaired in cIAP1/2-deficient mouse embryonic fibroblast (MEF) cell lines. Furthermore, it was shown that the E3 ligase activity of cIAPs, but not of TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. This result, together with the ability of HOIL-1 and HOIP to bind polyubiquitin chains of various linkage types, suggests that LUBAC is recruited to the TNF-RSC via cIAP-generated ubiquitin chains. It was also found that LUBAC enhances NEMO interaction with the TNF-RSC, that it stabilises this protein complex and that LUBAC is required for efficient TNF-induced activation of NF-kB and JNK, resulting in apoptosis inhibition. Finally, it is shown in this thesis that the catalytic activity of LUBAC is required for stabilisation of the TNF-RSC, thereby adding a novel form of ubiquitin linkage to TNF signalling. The identification of HOIL-1 and HOIP as functional constituents of the TNF-RSC provides evidence that LUBAC is an important regulator at the apex of TNF-induced signalling cascades and increases the combinatorial complexity of ubiquitin modifications within this receptor complex