TNF Knockout Cell Line (HEK 293)

Tumor necrosis factor (TNF)-induced receptor-interacting serine/threonine protein kinase 1 (RIPK1)-mediated cell death, including apoptosis and necroptosis, is increasingly recognized as a major driver of inflammatory diseases. Cell death checkpoints normally suppress RIPK1 kinase to safeguard the organism from its detrimental consequences. However, the mechanisms licensing RIPK1 kinase activity when a protective checkpoint is disabled remain unclear. Here, we identified S-palmitoylation as a licensing modification for RIPK1 kinase. TNF induces RIPK1 palmitoylation, mediated by DHHC5 and dependent on K63-linked ubiquitination of RIPK1, which enhances RIPK1 kinase activity by promoting the homo-interaction of its kinase domain and promotes cell death upon cell death checkpoint blockade. Furthermore, DHHC5 is amplified by fatty acid in the livers of mice with metabolic dysfunction-associated steatohepatitis, contributing to increased RIPK1 cytotoxicity observed in this condition. Our findings reveal that ubiquitination-dependent palmitoylation licenses RIPK1 kinase activity to induce downstream cell death signaling and suggest RIPK1 palmitoylation as a feasible target for inflammatory diseases.

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.