Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death

Area of Science:

• Cellular and Molecular Biology
• Immunology
• Genetics

Background:

• Receptor-interacting protein 1 (RIPK1) is crucial for inflammatory signaling and cell death pathways.
• RIPK1 ubiquitination regulates TNF-TNFR1 signaling, NF-κB, and MAPK activation.
• Specific lysine residues, including K376 and K115, are key sites for RIPK1 ubiquitination.

Purpose of the Study:

• To investigate the functional role of RIPK1 ubiquitination at K376 and K115 in vivo.
• To determine the impact of impaired RIPK1 ubiquitination on TNF-induced signaling and cell death.
• To elucidate the mechanisms underlying RIPK1-mediated embryonic lethality.

Main Methods:

• Generation and analysis of RIPK1 K376R and K115R mutant mice.
• Administration of RIPK1 inhibitor GNE684 during pregnancy.
• Genetic deletion of TNFR1, caspase-8, and RIPK3 in RIPK1 mutant mice.
• Assessment of TNF-induced hypothermia and lethality in adult mice.
• Analysis of RIPK1 ubiquitination status and signaling complex assembly.

Main Results:

• Mutation of RIPK1 K376 to arginine (K376R) in mice leads to embryonic lethality due to RIPK1 kinase-dependent cell death.
• RIPK1 K376R mutation diminishes various RIPK1 ubiquitination linkages and promotes death-inducing complex assembly.
• Embryonic lethality in Ripk1^K376R/K376R mice is rescued by TNFR1 loss or combined caspase-8/RIPK3 deletion.
• Adult Ripk1^K376R/cko mice are highly sensitive to TNF-induced lethality.
• Mutation at K115 (K115R) did not significantly affect RIPK1 ubiquitination or TNF signaling.

Conclusions:

• Selective impairment of RIPK1 ubiquitination at K376 lowers the threshold for RIPK1 activation by TNF.
• This dysregulation results in excessive cell death and embryonic lethality.
• RIPK1 ubiquitination is critical for maintaining the stability of signaling complexes that activate NF-κB and MAPK pathways.