Characterisation of SHARPIN as a third component of the linear ubiquitin chain assembly complex (LUBAC)

Abstract

Tumour necrosis factor (TNF) is an important cytokine with great physiological relevance and effects ranging from pro-inflammatory to immuno-regulatory functions. On a cellular level, it induces signalling processes by crosslinking its receptors and by initiating the formation of an intracellular, multi-protein receptor-signalling complex (RSC). Investigation of the TNF-RSC by modified tandem affinity purification (moTAP) and mass spectrometry revealed the presence of three novel components in this complex: heme-oxidised IRP2 ubiquitin ligase-1 (HOIL-1), HOIL-1-interacting protein (HOIP) and SHANK-associated RH-domain-interacting protein (SHARPIN). Previous studies showed that HOIL-1 and HOIP form an E3-complex that mediates the generation of linearly linked ubiquitin chains and is hence referred to as linear ubiquitin chain assembly complex (LUBAC). Identification of peptides specific for SHARPIN in the mass spectrometric analysis of the native TNF-RSC together with its sequence similarity to HOIL-1 raised the questions whether SHARPIN contributes functionally to TNF-signalling and/or the E3-activity of LUBAC. In this thesis, it could be shown that all three proteins are specifically recruited to the TNF-RSC in a cIAP1/2-dependent manner. As SHARPIN, HOIL-1 and HOIP can bind ubiquitin chains this suggests that the three proteins are recruited via cIAP1/2-generated ubiquitin chains. In addition, HOIP is required for presence of SHARPIN and HOIL-1 in the TNF-RSC. This, together with the finding that these three factors form a stimulation-independent protein complex in the cytosol, indicates that LUBAC is recruited to the TNF-RSC as a tripartite complex via its central component HOIP. In-vitro ubiquitination assays showed that SHARPIN is not only a physical but also a functional component of LUBAC. HOIP can generate ubiquitin chains when combined with either SHARPIN, HOIL-1 or both and was shown to exclusively generate linear linkages via a HECT-like mechanism. NEMO was identified as a common target of all possible LUBAC-combinations in vitro and, in line with this, the activity of LUBAC is required for full activation of NF-B following TNF stimulation. The results obtained in this thesis identify SHARPIN as a third component of LUBAC, an E3-complex that is specifically recruited to the TNF-RSC and regulates TNF signalling by modifying specific target proteins with linearly linked ubiquitin chains.