Human ZBP1 is a potent inducer of cell death through mechanisms divergent from mouse ZBP1.

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.