The Z-nucleic acid sensor ZBP1 in health and disease

Abstract

Nucleic acid sensing is a central process in the immune system, with far-reaching roles in antiviral defense, autoinflammation, and cancer. Z-DNA binding protein 1 (ZBP1) is a sensor for double-stranded DNA and RNA helices in the unusual left-handed Z conformation termed Z-DNA and Z-RNA. Recent research established ZBP1 as a key upstream regulator of cell death and proinflammatory signaling. Recognition of Z-DNA/RNA by ZBP1 promotes host resistance to viral infection but can also drive detrimental autoinflammation. Additionally, ZBP1 has interesting roles in cancer and other disease settings and is emerging as an attractive target for therapy.

Figure 1.

ZBP1 domain structure and signaling. (A) Schematic overview of the domain architectures of the two major human and mouse isoforms of ZBP1 detectable by Western blot. UniProt identifiers are shown below each structure. Humans express a second isoform termed ZBP1-S, which lacks exon 2 encoding the first Zα domain, while mice express a second isoform only encompassing the two Zα domains. The role of these isoforms remains mostly unknown; human ZBP1-S is thought to induce a MAVS-dependent type I IFN response following recognition of TERRA transcripts (see “The role of ZBP1 in cancer”). (B) Top: AlphaFold prediction of the structure of human ZBP1. Both Zα domains form a winged helix-turn-helix structure that enables them to specifically bind to Z-RNA/DNA. The β-sheets of the three consecutive RHIMs stack in an amyloidal structure on top of each other. Bottom: Z-DNA/Zα2 crystal structure (PDB 3EYI). Two Zα2 domains bind in an antiparallel fashion to Z-DNA with a 5 bp footprint. Tyr¹⁴⁵ located in the third α-helix of the Zα domain forms the only base contact with syn-dG (both shown in green) through a CH-π interaction. (C) Schematic of the different signaling outcomes of ZBP1 activation. ZBP1 may interact with Z-DNA/RNA in two manners. Due to the chemical equilibrium, a fraction of right-handed dsDNA/RNA adopts the Z-conformation. These molecules are then “trapped” in the left-handed Z conformation through conformational selection by the Zα domains of ZBP1. Alternatively, Z-prone nucleic acids may be actively “pushed” into the Z conformation by ZBP1’s Zα domains (induced fit). Activated ZBP1 then induces downstream signaling via its RHIMs. During NF-κB activation, the first RHIM of ZBP1 (RHIM-A) mediates binding to RIPK1 and RIPK3 through homotypic RHIM interactions. Both K63- and M1-linked ubiquitin chains are then attached to ZBP1 and RIPK1 by the K63-specific ubiquitin E3 ligases cIAP1 and cIAP2 and the linear ubiquitin chain assembly complex (LUBAC), which mediates M1 ubiquitination. These ubiquitin chains then recruit the TAK1 and IKK kinase complexes, which activate the NF-κB transcription factor resulting in inflammatory gene expression. ZBP1 has also been reported to activate the IRF3 transcription factor via TBK1 to induce type I IFN expression. The detailed mechanisms that are involved in this process remain to be described. The ZBP1/RIPK3/RIPK1 complex can also induce apoptosis after recruitment and activation of caspase-8 via FADD. Like TNF signaling, this may occur when ubiquitination of the ZBP1/RIPK3/RIPK1 complex is perturbed or when the expression of anti-apoptotic proteins such as cFLIP is downregulated. When caspase-8 activation is inhibited, ZBP1 activation results in the formation of a necrosome whereby RIPK3 phosphorylates and activates the pore-forming protein MLKL, resulting in necroptosis. At least in mouse cells, this occurs independently of RIPK1. In macrophages, ZBP1 stimulates activation of the NLRP3 inflammasome through a mechanism that has not yet been clearly defined, resulting in activation of caspase-1, which cleaves and activates the pore-forming protein GSDMD, resulting in pyroptosis. Figures of DNA, RNA, and dying cells were created with BioRender.com.

Figure 2.

Negative regulation of ZBP1 activation. (A) (i) Apoptosis and necroptosis induction downstream of mouse ZBP1 is inhibited by the RHIM-B/C containing C-terminus. The mechanism by which this occurs is not yet clear. (ii) Recruitment and activation of caspase-8 by RIPK1 and FADD to active ZBP1 prevents necroptosis. Caspase-8 proteolytically cleaves mouse RIPK3 after Asp³³³ thereby releasing its kinase domain from the signaling complex. A similar mechanism may be involved in restraining ZBP1-mediated necroptosis in human cells. (B) (iii, left) Trimethylation (me3) of histone H3 Lys9 by SETDB1 at loci coding for endogenous retroviruses (ERVs) suppresses ERV transcription. (iii, right) In the absence of SETDB1, overlapping sense and antisense transcripts are transcribed from the bidirectional long-terminal repeat (LTR) promotors of ERVs. These transcripts form dsRNA, which may adopt the Z-conformation and activate ZBP1 in the cytosol. (iv, left) Sequestration of Z-RNA by the Zα domain of ADAR1 prevents ZBP1 activation. Alternatively, mutual binding of ADAR1 and ZBP1 to Z-RNA prevents RIPK3 recruitment to ZBP1. ADAR1 binds Z-RNA formed by foldback of IR-Alus found in the 3′ UTRs of many genes or short complementary sequences within the 3′ UTRs of IFN-stimulated genes (ISGs). The Zα domain of ADAR1 further enhances adenosine-to-inosine (A-to-I) editing of IR-Alus, which prevents recognition of these structures by ZBP1. (iv, right) Loss of ADAR1 function results in the accumulation of unedited (Z-form) dsRNA inside the cytosol and the recognition of Z-RNAs by ZBP1, resulting in its activation. Figures of DNA and RNA were created with BioRender.com.