RIPK1 inhibits ZBP1-driven necroptosis during development
Kim Newton1, Katherine E Wickliffe1, Allie Maltzman1
1Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA.
Abstract:
Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
Insights
Receptor-interacting protein kinase 1 (RIPK1) normally prevents Z-DNA binding protein 1 (ZBP1) from activating RIPK3-dependent necroptosis. Loss of RIPK1
Area of Science:
- Cellular biology
- Immunology
- Molecular mechanisms of cell death
Background:
- Receptor-interacting protein kinase 1 (RIPK1) is crucial for cell survival, as RIPK1 deficiency leads to perinatal lethality due to aberrant apoptosis and necroptosis.
- While catalytically inactive RIPK1 mutants allow survival, suggesting a scaffold function, the precise role of RIPK1 in regulating cell death pathways remains incompletely understood.
- The RIP homotypic interaction motif (RHIM) is a key domain in RIPK1 involved in protein-protein interactions within cell death signaling.
Purpose of the Study:
- To investigate the role of the RIP homotypic interaction motif (RHIM) of Receptor-interacting protein kinase 1 (RIPK1) in preventing programmed cell death during development.
- To elucidate the interaction between RIPK1, Z-DNA binding protein 1 (ZBP1), and RIPK3 in the context of necroptosis regulation.
- To determine if ZBP1-mediated RIPK3 activation contributes to the lethality observed in mice with impaired RIPK1 function.
Main Methods:
- Generation and analysis of Ripk1RHIM/RHIM mice with mutated RIPK1 RHIM residues.
- Assessment of necroptosis markers (RIPK3 autophosphorylation) and lethality in genetically modified mouse models (e.g., Ripk3 knockout, MLKL knockout).
- Co-immunoprecipitation assays to study protein-protein interactions between ZBP1 and RIPK3 in various genetic backgrounds.
Main Results:
- Mice expressing RIPK1 with a mutated RHIM (Ripk1RHIM/RHIM) exhibited perinatal lethality and RIPK3 autophosphorylation, indicating necroptosis.
- Blocking necroptosis through genetic deficiency of RIPK3 or MLKL, or by using catalytically inactive RIPK3, rescued lethality in Ripk1RHIM/RHIM mice.
- Loss of Z-DNA binding protein 1 (ZBP1) also prevented lethality in Ripk1RHIM/RHIM mice, and ZBP1 interacted with RIPK3 in the absence of functional RIPK1 and MLKL.
Conclusions:
- The RHIM domain of RIPK1 acts as a critical brake, preventing ZBP1 from inappropriately engaging RIPK3 and initiating necroptosis during development.
- ZBP1-RIPK3-MLKL-dependent necroptosis is a key pathway that must be suppressed by RIPK1 for normal embryonic and perinatal survival.
- These findings reveal a novel inhibitory role for the RIPK1 RHIM in safeguarding development by controlling ZBP1-mediated necroptotic signaling.
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