RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1^R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.

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.

Acute kidney injury (AKI) is one of the most common and lethal complications in severely burned patients. Its clinical management has long been challenged by a dilemma: high incidence and mortality rates coexist with the absence of targeted therapies, largely due to an incomplete understanding of the molecular networks that drive “burn-to-kidney” injury. Here, we report the renal proteomic landscape of burn-induced AKI and identify ferroptosis as a critical mechanism underlying its pathogenesis. Using a murine model of burn-induced AKI that recapitulates elevated serum creatinine and urea levels as well as tubular necrosis, quantitative proteomics revealed widespread protein changes, with upregulated proteins enriched in iron transport and ferroptosis‑related pathways. Specifically, ferroptosis-associated proteins ACSL4, FTH1, FTL1, and MT1/2 were upregulated and highly correlated with the tubular injury marker KIM1. Importantly, pharmacological inhibition of ferroptosis effectively attenuated burn-induced AKI, as evidenced by reduced serum biomarkers, decreased tubular necrosis, and improved survival. Together, these findings unveil a burn‑specific ferroptosis‑related molecular signature and provide a rationale for targeted intervention in burn‑induced AKI.

Regulation of enhancer activity plays a pivotal role in governing gene expression and cellular behaviors. However, the precise mechanisms underlying dynamic control of active enhancers remain incompletely defined. Here, we demonstrate that the nuclear receptor estrogen-related receptor α (ERRα) forms a functional complex with the H3K4me3-specific demethylase KDM5C to co-occupy a large set of active enhancers, including the locus of STING. In breast cancer cells, ERRα depletion induces STING enhancer hyperactivation, evidenced by H3K4me3 deposition, decreased H3K4me1, and increased enhancer RNA (eRNA) transcription. Accordingly, depletion of ERRα leads to further activation of STING gene transcription and TBK1-IRF3 pathway, accompanied by increased type I interferon (IFN) and IFN-stimulated gene (ISG) expression, as confirmed by transcriptomic analysis. Notably, depleting ERRα markedly attenuates breast tumor cell growth in vitro and in vivo, and our in vitro evidence indicates this occurs in part through activating STING signaling. These findings establish that the ERRα-KDM5C serves as a critical checkpoint for STING enhancer activity, revealing a regulatory mechanism of STING enhancer activity in breast tumor progression.