Medical Biology - Theses

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Author: Tanzer, Maria × End date: 2019 × Start date: 2011 × Type: PhD thesis × Subject: apoptosis × Subject: necroptosis ×

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    Investigation of cell death pathways in response to TNF and IFNγ
    Tanzer, Maria ( 2017)
    During my PhD I investigated the regulation of the TNF and IFNγ signalling pathways and their ability to induce cell death. IFNγ is a critical cytokine in the immune response against viral and intracellular bacterial infections. It has also been associated with auto-inflammatory and auto-immune disorders (Pollard et al., 2013; Zhang, 2007), where it was found upregulated together with other pro-inflammatory cytokines like TNF (Ohmori et al., 1997). TNF signalling and the mechanism of cell death induction downstream of the TNF receptor complex has been investigated in detail over the past 4 decades. Although IFNγ was first described 50 years ago, and before TNF, significantly fewer IFNγ signalling components have been discovered compared to the TNF signalling complex. Nevertheless, both cytokines induce equally potent and potentially dangerous systemic responses at low concentrations and must be tightly regulated. I therefore hypothesised that additional IFNγ signalling regulators must exist. In order to discover such novel regulators of the IFNγ signalling pathway I enriched for the IFNγ receptor and identified binding partners using mass spectrometry. Using this approach I identified SPTLC1 and 2, which are two subunits forming the serine palmitoyltransferase, directly interacting with the IFNγ receptor chain 2 (IFNGR2) constitutively. Weak interaction between SPTLC1/2 and IFNGR1, however, was only detected upon IFNGR complex formation induced by IFNγ stimulation suggesting that IFNGR1 interacts with SPTLC1/2 indirectly via IFNGR2. SPTLC2 deficient single cell mouse dermal fibroblast showed either normal or increased phosphorylation of STAT1 upon IFNγ stimulation and lack of SPTLC2 had no impact on transcription of classical IFN target genes. Secondly, I investigated the mechanism of cell death induced by IFN in combination with Smac-mimetics, a group of small molecule inhibitors of the inhibitor of apoptosis proteins (IAPs), which have been heavily investigated in context of TNF signalling. Previous studies revealed that inhibition of IAPs renders cells sensitive to TNF induced cell death, which is primarily apoptosis mediated by caspase-8. However, inhibition of caspase-8 by caspase inhibitors triggers an alternative cell death pathway called necroptosis. Here I found that the combination of IFN/Smac-mimetic had a similar impact on survival and, more precisely, induced RIPK3 dependent caspase-8 mediated apoptosis in mouse dermal fibroblasts. Surprisingly, IFN/Smac-mimetic induced killing in HT29 cells was not blocked by deleting caspase-8 and effectors of the necroptotic pathway like RIPK3 and MLKL. In contrast, deficiency of RIPK1 largely protected cells from IFN induced cell death, indicating that a novel form of RIPK1 dependent cell death was being induced. In trying to discover the mechanism we observed that caspase-10 was significantly upregulated by IFN and activated by IFN/Smac-mimetic treatment. HT29 cells deficient for caspase-10, caspase-8 and either MLKL or RIPK3 were completely resistant to IFN/Smac-mimetic revealing an important role for caspase-10 in IFN/Smac-mimetic induced killing. Thirdly I focused on the activation and function of MLKL, the most downstream member of the necroptotic pathway known. Necroptosis has been best studied downstream of the TNF signalling complex, upon IAP and caspase inhibition. We and others propose a model where phosphorylation of the MLKL pseudokinase domain by RIPK3 triggers a molecular switch, leading to exposure of MLKL’s N-terminal four-helix bundle domain, its oligomerisation, membrane translocation, and ultimately cell death. We additionally identified novel phosphorylation sites S158, S228, S248. By mutating these sites and overexpressing phosphomimetic and -ablating MLKL mutants in Mlkl-/- or Ripk3-/-/Mlkl-/- deficient murine fibroblasts I demonstrated that these sites influence MLKL activity and discovered a potential inhibitory effect of RIPK3 on cell death induced by MLKL. Finally, I examined the evolutionarily conservation of the necroptosis inducing activity of MLKL by analysing the function of MLKL orthologs. While the intrinsic ability to lyse membranes, which was tested in liposome assays, is highly conserved, several MLKL orthologs including human MLKL failed to induce cell death when expressed in murine fibroblasts. This suggests the presence of additional poorly conserved, species-specific factors that inhibit or activate MLKL.