Mutations of theADAR1gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi–Goutières syndrome and bilateral striatal necrosis^1–3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA^4,5. HemizygousADAR1mutations in the Zα domain cause type I IFN-mediated pathologies in humans^2,3and mice^6–8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains⁹, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1^mZα/–mice).Adar1^mZα/–mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology inAdar1^mZα/–mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused byADAR1mutations.

Z-DNA binding protein 1 (ZBP1) has important functions in anti-viral immunity and in the regulation of inflammatory responses. ZBP1 induces necroptosis by directly engaging and activating RIPK3, however, the mechanisms by which ZBP1 induces inflammation and in particular the role of RIPK1 and the contribution of cell death-independent signaling remain elusive. Here we show that ZBP1 causes skin inflammation by inducing RIPK3-mediated necroptosis and RIPK1-caspase-8-mediated apoptosis in keratinocytes. ZBP1 induced TNFR1-independent skin inflammation in mice with epidermis-specific ablation of FADD by triggering keratinocyte necroptosis. Moreover, transgenic expression of C-terminally truncated constitutively active ZBP1 (ZBP1ca) in mouse epidermis caused skin inflammation that was only partially inhibited by abrogation of RIPK3-MLKL-dependent necroptosis and fully prevented by combined deficiency in MLKL and caspase-8. Importantly, ZBP1ca induced caspase-8-mediated skin inflammation by RHIM-dependent but kinase activity-independent RIPK1 signaling. Furthermore, ZBP1ca-induced inflammatory cytokine production in the skin was completely prevented by combined inhibition of apoptosis and necroptosis arguing against a cell death-independent pro-inflammatory function of ZBP1. Collectively, these results showed that ZBP1 induces inflammation by activating necroptosis and RIPK1 kinase activity-independent apoptosis.

Z-DNA-binding protein 1 (ZBP1) senses Z-form nucleic acids to trigger cell death and inflammation via RHIM domain-mediated interactions with RIPK1 and RIPK3. Here we show that compared to mouse ZBP1 (mZBP1), human ZBP1 (hZBP1) possesses a heightened sensitivity for inducing cell death in cells across different species and potent tumor-killing effects in vivo. In contrast to mZBP1, which signals primarily through RIPK3, hZBP1-induced cell death depends on RIPK1 in a RIPK3-independent manner. Specifically, while the scaffold function of RIPK1 is required for hZBP1-mediated apoptosis, its kinase activity is indispensable for the execution of necroptosis. Unlike mZBP1, which primarily uses only its RHIM1 domain, hZBP1 requires all three RHIM domains (RHIM1, RHIM2, and RHIM3) to trigger cell death. We further identify the C-terminal RHIM2 and RHIM3 regions as the key determinant that confers hZBP1 high sensitivity and confirm that endogenous hZBP1 promotes RIPK1-dependent cell death under pathological conditions. Together, our findings reveal an intrinsic mechanistic divergence between human and murine ZBP1 signaling and highlight the limitations of translating preclinical findings in animal models to human therapeutic strategies targeting this pathway.

Z-DNA binding protein 1 (ZBP1), a sensor of Z-form nucleic acids, plays a crucial role in cell death and inflammation. While its functions in infection and development are well-established, its involvement in environmental stress responses remains largely unexplored. In this study, we uncovered novel mechanisms by which ZBP1 mediates cell death under arsenic and hyperosmotic stress. We demonstrate that ZBP1 initiates necroptosis during the early stages of these stresses. As the stress persists, the cell death mode evolves, shifting towards apoptosis and pyroptosis in later stages. This transition is particularly pronounced when the necroptotic pathway is compromised. Interestingly, despite previous implications of stress granules (SGs) in arsenic-dependent cell death, we found that neither SGs formation inhibitors nor the ablation of SGs components significantly impacts cell death under arsenic or hyperosmotic stress. This suggests that environmental stress-induced necroptosis operates largely independent of SGs formation. Employing genome-wide CRISPR/Cas9 library screening, we identified that ZBP1-dependent cell death in response to arsenic is primarily driven by reactive oxygen species and the KEAP1-NRF2 signaling pathway. Notably, ZBP1 KO mice demonstrated resistance to arsenic-induced cell death and tissue injury, further substantiating our findings. Our research provides important new insights into ZBP1's role in environmental stress-induced cell death, expanding our understanding beyond its established functions in infection and development. These findings offer potential implications for comprehending stress-related tissue injury mechanisms.