Regulation of programmed necrosis by CYLD cleavage.

DESCRIPTION (provided by applicant): Caspase-8 is an initiator caspase that is essential for apoptotic death triggered by TNFR1. However, there are suggestions that Caspase-8 may also have the opposite function of keeping cells alive but how Caspase-8 mediates a survival signal is not known. In preliminary studies, we found that Caspase-8 does this by cleaving the tumor suppressor CYLD at residue D215 resulting in CYLD's degradation. Because CYLD is required for programmed necrosis or necroptosis, its degradation has the effect of suppressing this form of cell death. CYLD is a deubiquitinase that specifically removes K63-linked, as well as linear M1-linked, polyubiquitin modifications from targets such as Ripk1 and Nemo. In mouse embryonic fibroblasts (MEFs), blocking CYLD cleavage by deleting Casp8 or mutating the cleavage site on CYLD (D215A) causes the cells to undergo necrotic death following TNF stimulation. When CYLD is degraded, there is enhanced ubiquitination of Ripk1 favoring its association with Nemo to form a survival- signaling complex. When CYLD is retained, there is less ubiquitination of Ripk1 favoring its association with the Ripk3-containing necrosome complex. Based on these observations, we propose the hypothesis that caspase-mediated degradation of CYLD is the key event in keeping cells alive after TNF stimulation because this suppresses necrosis. We will test this hypothesis by: (1) characterizing the Caspase-8 complex involved in cleaving CYLD; (2) identifying and characterizing the role of CYLD's targets during necrosis; (3) examining the in vivo consequence of Caspase-8-mediated cleavage of CYLD by generating CYLD-D215A knock-in mice. A clearer understanding of how necrosis is regulated, and whether its dysregulation could lead to inflammation, could indicate whether modulating necrotic death may be of potential therapeutic value in inflammatory disorders such as psoriasis, rheumatoid arthritis and inflammatory bowel diseases where TNF is known to be involved.