Z-DNA-binding protein 1, also known as DNA-dependent activator of IFN-regulatory factors (DAI) and DLM-1, is a protein that in humans is encoded by the ZBP1 gene. ZBP1 is also an abbreviation for chicken or rat β-actin zipcode-binding protein 1, a homolog of the human insulin-like growth factor 2 mRNA-binding protein 1 (IMP-1) and murine CRD-BP, the proteins involved in mRNA transport (RNA-binding proteins, RBPs).

History

ZBP1 was first identified as an interferon-inducible Z-NA binding protein, but its specific functions remained unclear for many years. It was initially thought to be a cytosolic DNA sensor. However, the generation of Zbp1-deficient mice revealed that these mice responded normally to DNA and DNA virus infections, producing normal levels of interferon. Further insights came with the discovery of ZBP1's receptor-interacting protein homotypic interaction motif (RHIM) domains, which mediate interactions with other proteins. Experiments showed that ZBP1 interacts with RIPK1 and RIPK3 through these RHIM domains. This interaction hinted at ZBP1's involvement in cell death, especially given the role of RIPK proteins in cell death pathways. The role of ZBP1 as an innate immune sensor became more evident with the discovery that it regulates NLRP3 inflammasome activation and inflammatory cell death, PANoptosis, during influenza A virus infection. ZBP1-deficient mice showed impaired activation of inflammasome components, such as caspase-1, and reduced levels of IL-1β and IL-18, highlighting its critical role in antiviral defense.

Structure

ZBP1 has several key domains that contribute to its function. At the N-terminus, it has Z-nucleic acid (Z-NA) binding domains, known as Zα1 and Zα2. Both Zα domains have a winged helix-turn-helix structure which allows them to bind to Z-RNA/DNA. The intermediate region of ZBP1 contains two receptor-interacting protein homotypic interaction motif (RHIM) domains, RHIM1 and RHIM2, which facilitate interactions with other RHIM domain-containing proteins. The C-terminal region of ZBP1 contains a signal domain (SD), which is crucial for triggering an interferon response.

Function and role in disease

ZBP1 was discovered as an innate immune sensor of influenza A virus that forms the ZBP1-PANoptosome to activate the NLRP3 inflammasome and drive cell death, PANoptosis. PANoptosis is a prominent innate immune, inflammatory, and lytic cell death pathway initiated by innate immune sensors and driven by caspases and receptor-interacting protein kinases (RIPKs) through PANoptosomes. PANoptosomes are multi-protein complexes assembled by germline-encoded pattern-recognition receptor(s) (PRRs) (innate immune sensor(s)) in response to pathogens, including bacterial, viral, and fungal infections, as well as pathogen-associated molecular patterns, damage-associated molecular patterns, cytokines, and homeostatic changes during infections, inflammatory conditions, and cancer. Following the discoveries with IAV, the ZBP1-PANoptosome was also found to play a role in pathogenic inflammation in response to IFN therapy during coronavirus infection. In contrast, the ZBP1-PANoptosome can have therapeutic benefit in murine tumor models, where IFN and nuclear export inhibitor treatment modulates the ZBP1-ADAR1 pathway and drives ZBP1-PANoptosome formation to regress tumors. Recent research has identified curaxin (CBL0137), a small molecule inhibitor, to effectively induce ZBP1-mediated cell death in cancer-associated fibroblasts and effectively reverse immune checkpoint blockade (ICB) resistance in mouse models of melanoma. ZBP1 is also a key component of the absent in melanoma 2 (AIM2)-PANoptosome, which includes the AIM2 inflammasome, and assembles in response to Francisella novicida and herpes simplex virus 1 (HSV1) infections. ZBP1 has also been extensively implicated in other viral infections, including coronaviruses, cytomegalovirus (CMV), vaccinia (VACV), varicella-zoster virus, zika virus (ZIKV), and others. Furthermore, ZBP1 also induces PANoptosis during Candida albicans and Aspergillus fumigatus infections.

Z-NA binding

ZBP1 is proposed to be a Z-DNA binding protein. Z-DNA formation is a dynamic process, largely controlled by the amount of supercoiling. ZBP1 recognizes DNA in the cytoplasm as an antiviral mechanism. Viral life cycles often include steps where DNA is exposed in the cytoplasm. DNA is normally contained in the nucleus of a cell, and therefore cells use proteins like ZBP1 as an indicator of a viral infection. Once ZBP1 is activated, it increases the production of antiviral cytokines such as interferon beta. DLM1 then binds to cytosolic Viral DNA using two Z-DNA-binding domains (Zα and Zβ) at its N-terminus along with a DNA binding domain (D3). The role of ZBP1 in DNA sensing has been questioned. It has been found to sense Influenza A Virus (IAV) infection and induce cell death. Since DNA is not synthesized in any stage of IAV life cycle, DNA sensing playing a role in this context is unlikely. However, recent investigation has found that ZBP1 is capable of sensing Z-form RNAs produced during IAV infection, cumulating in a form of caspase independent, inflammatory cell death called necroptosis. A follow-up study identified that ZBP1 senses the IAV ribonucleoprotein complex to induce cell death. A more recent study has identified transcription factor IRF1 as the upstream regulator of ZBP1 expression.