P7: Proteases in cell survival

Pro-survival signalling pathways involving caspase-8 (PI Ulrich Maurer).

Caspases are proteases with defined roles in the execution of apoptosis and the processing and release of inflammatory cytokines. Recently, a pro-survival role of caspase-8, by the prevention of programmed necrosis, was identified. When cells are stimulated with the inflammatory cytokine TNF, a protein complex is assembled which mediates the protease activity of caspase-8, but this activity of caspase-8 does not induce apoptosis. While some substrates of this activity have been defined, we hypothesize that novel substrates with unknown functions remain to be identified. We have found proteins, which are recruited to the TNFR1 signalling complex, can be cleaved by caspase-8 in vitro. This project aims at i.) the characterization of current caspase-8 substrate candidates in the lab and  ii.) the identification of novel caspase-8 substrates with non-apoptotic functions.

ProtPath offers two topics for doctoral work on caspase-8:

1.  The role of the proteolytic cleavage of substrate candidates of caspase-8

To analyse the relevance of the cleavage of substrate candidates, we will first identify the respective cleavage sites by generating mutants of the candidate cleavage sites. We will test those in in vitro cleavage assays. The mutants will then be tested for resistance against proteolysis in cells stimulated with TNF. By introduction of the cleavage-deficient mutants in into the respective knock-out cells, or CRISPR/Cas9-mediated knock-in cells, we will test the relevance of the cleavage the candidate for TNF-induced inflammation. We will explore whether the absence of the cleavage of candidate proteins affects poly-ubiquitylation or phosphorylation events in the TNFR1 signalling complex, the formation or stability of TNFR1 complexes, or pro-inflammatory cytokine expression. We will also introduce the N- and C-terminal cleavage products individually in cells and likewise analyse their possible (i.e. dominant negative) role for cell death and/or pro-inflammatory cytokine signalling.

2. Identification of novel caspase-8 substrates by N-TAILS

We aim at defining novel targets of the non-apoptotic activity of caspase-8, employing a global proteomic approach. Cells deficient for programmed necrosis will be stimulated with TNF in the presence or absence of caspase inhibitor, followed by mass-spectrometry analysis by N-TAILS. Candidate targets will be verified by in vitro cleavage experiments, and the caspase-8 cleavage sites will be identified. Cleavage-deficient mutants of the candidates will be generated, and the capability to modulate inflammation or cell death of those candidate proteins will be assessed.