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Biochemical and functional characterisation of the Linear Ubiquitin Chain Assembly Complex (LUBAC) as a component of the TNF-Receptor 1 signalling complex
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
XMILE:An XML-based approach for programmable networks
In this paper we describe an XML-based platform for dynamic active node policy updates. XML supports the definitionof specific policy languages, their extension to satisfy new needs and the management of deployed policies on differentactive nodes. We show an example of the management of router packet forwarding policies where the XML policiesthat drive the packet routing are updated at run-time on the active nodes depending on the network status. The platformdecouples policy management, which is handled through XML interpretation, from packet forwarding that, forperformance reasons has to be implemented in more efficient languages
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