Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Polyamine metabolism controls B-to-Z DNA transition to orchestrate DNA sensor cGAS activity.

Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.

Noncanonical DNA structures are drivers of genome evolution.

In addition to the canonical right-handed double helix, other DNA structures, termed 'non-B DNA', can form in the genomes across the tree of life. Non-B DNA regulates multiple cellular processes, including replication and transcription, yet its presence is associated with elevated mutagenicity and genome instability. These discordant cellular roles fuel the enormous potential of non-B DNA to drive genomic and phenotypic evolution. Here we discuss recent studies establishing non-B DNA structures as novel functional elements subject to natural selection, affecting evolution of transposable elements (TEs), and specifying centromeres. By highlighting the contributions of non-B DNA to repeated evolution and adaptation to changing environments, we conclude that evolutionary analyses should include a perspective of not only DNA sequence, but also its structure.

Flipons and small RNAs accentuate the asymmetries of pervasive transcription by the reset and sequence-specific microcoding of promoter conformation.

The role of alternate DNA conformations such as Z-DNA in the regulation of transcription is currently underappreciated. These structures are encoded by sequences called flipons, many of which are enriched in promoter and enhancer regions. Through a change in their conformation, flipons provide a tunable mechanism to mechanically reset promoters for the next round of transcription. They act as actuators that capture and release energy to ensure that the turnover of the proteins at promoters is optimized to cell state. Likewise, the single-stranded DNA formed as flipons cycle facilitates the docking of RNAs that are able to microcode promoter conformations and canalize the pervasive transcription commonly observed in metazoan genomes. The strand-specific nature of the interaction between RNA and DNA likely accounts for the known asymmetry of epigenetic marks present on the histone tetramers that pair to form nucleosomes. The role of these supercoil-dependent processes in promoter choice and transcriptional interference is reviewed. The evolutionary implications are examined: the resilience and canalization of flipon-dependent gene regulation is contrasted with the rapid adaptation enabled by the spread of flipon repeats throughout the genome. Overall, the current findings underscore the important role of flipons in modulating the readout of genetic information and how little we know about their biology.

The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells.

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

Inflammatory cell death PANoptosis is induced by the anti-cancer curaxin CBL0137 via eliciting the assembly of ZBP1-associated PANoptosome.

OBJECTIVE: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. METHODS: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. RESULTS: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. CONCLUSIONS: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice.

Baicalin inhibits PANoptosis by blocking mitochondrial Z-DNA formation and ZBP1-PANoptosome assembly in macrophages.

PANoptosis is an emerging form of regulated cell death (RCD) characterized by simultaneous activation of pyroptotic, apoptotic, and necroptotic signaling that not only participates in pathologies of inflammatory diseases but also has a critical role against pathogenic infections. Targeting PANoptosis represents a promising therapeutic strategy for related inflammatory diseases, but identification of inhibitors for PANoptosis remains an unmet demand. Baicalin () is an active flavonoid isolated from Scutellaria baicalensis Georgi (Huangqin), a traditional Chinese medicinal herb used for heat-clearing and detoxifying. Numerous studies suggest that baicalin possesses inhibitory activities on various forms of RCD including apoptosis/secondary necrosis, pyroptosis, and necroptosis, thereby mitigating inflammatory responses. In this study we investigated the effects of baicalin on PANoptosis in macrophage cellular models. Primary macrophages (BMDMs) or J774A.1 macrophage cells were treated with 5Z-7-oxozeaenol (OXO, an inhibitor for TAK1) in combination with TNF-α or LPS. We showed that OXO plus TNF-α or LPS induced robust lytic cell death, which was dose-dependently inhibited by baicalin (50-200 µM). We demonstrated that PANoptosis induction was accompanied by overt mitochondrial injury, mitochondrial DNA (mtDNA) release and Z-DNA formation. Z-DNA was formed from cytosolic oxidized mtDNA. Both oxidized mtDNA and mitochondrial Z-DNA puncta were co-localized with the PANoptosome (including ZBP1, RIPK3, ASC, and caspase-8), a platform for mediating PANoptosis. Intriguingly, baicalin not only prevented mitochondrial injury but also blocked mtDNA release, Z-DNA formation and PANoptosome assembly. Knockdown of ZBP1 markedly decreased PANoptotic cell death. In a mouse model of hemophagocytic lymphohistiocytosis (HLH), administration of baicalin (200 mg/kg, i.g., for 4 times) significantly mitigated lung and liver injury and reduced levels of serum TNF-α and IFN-γ, concomitant with decreased levels of PANoptosis hallmarks in these organs. Baicalin also abrogated the hallmarks of PANoptosis in liver-resident macrophages (Kupffer cells) in HLH mice. Collectively, our results demonstrate that baicalin inhibits PANoptosis in macrophages by blocking mitochondrial Z-DNA formation and ZBP1-PANoptosome assembly, thus conferring protection against inflammatory diseases. PANoptosis is a form of regulated cell death displaying simultaneous activation of pyroptotic, apoptotic, and necroptotic signaling. This study shows that induction of PANoptosis is linked to mitochondrial dysfunction and mitochondrial Z-DNA formation. Baicalin inhibits PANoptosis in macrophages in vitro via blocking mitochondrial dysfunction and the mitochondrial Z-DNA formation and thereby impeding the assembly of ZBP1-associated PANoptosome. In a mouse model of hemophagocytic lymphohistiocytosis (HLH), baicalin inhibits the activation of PANoptotic signaling in liver-resident macrophages (Kupffer cells) in vivo, thus mitigating systemic inflammation and multiple organ injury in mice.

Nucleosomes and flipons exchange energy to alter chromatin conformation, the readout of genomic information, and cell fate.

Alternative non-B-DNA conformations formed under physiological conditions by sequences called flipons include left-handed Z-DNA, three-stranded triplexes, and four-stranded i-motifs and quadruplexes. These conformations accumulate and release energy to enable the local assembly of cellular machines in a context specific manner. In these transactions, nucleosomes store power, serving like rechargeable batteries, while flipons smooth energy flows from source to sink by acting as capacitors or resistors. Here, I review the known biological roles for flipons. I present recent and unequivocal findings showing how innate immune responses are regulated by Z-flipons that identify endogenous RNAs as self. Evidence is also presented supporting important roles for other flipon classes. In these examples, the dynamic exchange of energy between flipons and nucleosomes enables rapid switching of genetic programs without altering flipon sequence. The increased phenotypic diversity enabled by flipons drives their natural selection, with adaptations evolving faster than is possible by codon mutation alone.

ZBP1 promotes hepatocyte pyroptosis in acute liver injury by regulating the PGAM5/ROS pathway.

INTRODUCTION AND OBJECTIVES: Acute liver injury (ALI) is characterized by massive hepatocyte death with high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the physiopathological processes of ALI, which can damage mitochondria and release NLRP3 inflammasome particles, causing systemic inflammatory responses. Z-DNA Binding Protein 1 (ZBP1) is a sensor that induces cell death. Here, we investigated whether ZBP1 participates in hepatocyte pyroptosis and explored the possible pathogenesis of ALI. MATERIALS AND METHODS: Hepatocyte pyrotosis was induced with lipopolysaccharide (LPS) and nigericin (Nig), and the expression of Zbp1 (ZBP1) was examined by western blot analysis and RT-qPCR. Further, we transfected AML-12 (LO2 and HepG2) cell lines with Zbp1 (ZBP1) siRNA. After ZBP1 was silenced, LDH release and flow cytometry were used to measure the cell death; Western blot analysis and RT-qPCR were used to detect the marker of NLRP3 inflammasome activation and pyroptosis. We also detected the expression of mitochondrial linear rupture marker phosphoglycerate mutase family member 5 (PGAM5) using western blot analysis and reactive oxygen species (ROS) using the DCFH-DA method. RESULTS: The expression of ZBP1 was up-regulated in LPS/Nig-induced hepatocytes. Si-Zbp1 (Si-ZBP1) inhibited NLRP3 inflammasome activation and pyroptosis in LPS/Nig-induced hepatocytes. Moreover, ZBP1 silencing inhibited the expression of PGAM5 by reducing ROS production. CONCLUSIONS: ZBP1 promotes hepatocellular pyroptosis by modulating mitochondrial damage, which facilitates the extracellular release of ROS.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

The Binding Properties of Antibodies to Z-DNA in the Sera of Normal Healthy Subjects.

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin.

A Compendium of G-Flipon Biological Functions That Have Experimental Validation.

As with all new fields of discovery, work on the biological role of G-quadruplexes (GQs) has produced a number of results that at first glance are quite baffling, sometimes because they do not fit well together, but mostly because they are different from commonly held expectations. Like other classes of flipons, those that form G-quadruplexes have a repeat sequence motif that enables the fold. The canonical DNA motif (G3N1-7)3G3, where N is any nucleotide and G is guanine, is a feature that is under active selection in avian and mammalian genomes. The involvement of G-flipons in genome maintenance traces back to the invertebrate Caenorhabditis elegans and to ancient DNA repair pathways. The role of GQs in transcription is supported by the observation that yeast Rap1 protein binds both B-DNA, in a sequence-specific manner, and GQs, in a structure-specific manner, through the same helix. Other sequence-specific transcription factors (TFs) also engage both conformations to actuate cellular transactions. Noncoding RNAs can also modulate GQ formation in a sequence-specific manner and engage the same cellular machinery as localized by TFs, linking the ancient RNA world with the modern protein world. The coevolution of noncoding RNAs and sequence-specific proteins is supported by studies of early embryonic development, where the transient formation of G-quadruplexes coordinates the epigenetic specification of cell fate.

Simple Repeats as Building Blocks for Genetic Computers.

Processing of RNA involves heterogeneous nuclear ribonucleoproteins. The simple sequence repeats (SSRs) they bind can also adopt alternative DNA structures, like Z DNA, triplexes, G quadruplexes, and I motifs. Those SSRs capable of switching conformation under physiological conditions (called flipons) are genetic elements that can encode alternative RNA processing by their effects on RNA processivity, most likely as DNA:RNA hybrids. Flipons are elements of a binary, instructive genetic code directing how genomic sequences are compiled into transcripts. The combinatorial nature of this code provides a rich set of options for creating genetic computers able to reproduce themselves and use a heritable and evolvable code to optimize survival. The underlying computational logic potentiates a diverse set of genetic programs that modify cis-mediated heritability and disease risk.

The expression of antibodies to Z-DNA in the blood of patients with systemic lupus erythematosus: Relationship to autoantibodies to B-DNA.

To explore the antibody response to Z-DNA, a DNA conformation with a zig-zag structure, blood of patients with systemic lupus erythematosus (SLE) and otherwise healthy individuals (NHS) were assayed by ELISA using brominated poly(dGdC), a synthetic Z-DNA antigen. These studies showed that SLE patients commonly express antibodies to Z-DNA; NHS also had binding in this assay. In SLE blood, levels of antibodies to Z-DNA were related to those to B-DNA using calf thymus DNA as a source of B-DNA; cross-reactivity was demonstrated by adsorption experiments using DNA cellulose. As shown by dissociation assays, antibody binding of SLE anti-Z-DNA is sensitive to the effects of ionic strength, suggesting electrostatic binding. Since Z-DNA structure can be found in bacterial DNA as well as bacterial biofilms, these findings suggest that, in SLE, anti-DNA antibody responses can result from stimulation by DNA of bacterial origin, with cross-reactivity leading to autoreactivity.

Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids.

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαßDAI), which harbours two Zα domains, and for a ZαßDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαßDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαßDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

Synchrotron radiation circular dichroism spectroscopy of oligonucleotides at millimolar concentrations.

Circular dichroism spectroscopy of nucleic acids has been traditionally performed at sample concentrations orders of magnitude lower than what occur in biological systems. While recent work from us demonstrated the flexibility of an adjustable sample cell that allowed for successful recording of CD spectra of an 18- and a 21-mer double stranded DNA sequences at around 1 mM, sample concentrations beyond 1 mM present a challenge for standard benchtop CD spectrometers. In the present work, the synchrotron radiation circular dichroism (SRCD) spectra were recorded for d(CG)9 and a mixed 18-mer double stranded DNA at 1, 5, and 10 mM in 100 mM or 4 M NaCl. SRCD of low molecular weight salmon DNA was also measured at a 10 mg/ml concentration. These results represent the first report of CD spectra of DNA samples measured at concentrations comparable to those found in the nucleus. The results suggest that dsDNA maintain very similar structures at concentrations up to tens of mg/ml, as evident by the very similar CD patterns in this concentration range. Furthermore, the SRCD allowed for the recording of CD patterns of DNA in the far UV region, which is not readily accessible by standard benchtop CD spectropolarimeters. These far UV signals appear to be quite characteristic of DNA structures and are sensitive to sample conditions.

Thermodynamic analysis of Zα domain-nucleic acid interactions.

DNA/RNA molecules adopting the left-handed conformation (Z-form) have been attributed with immunogenic properties. However, their biological role and importance have been a topic of debate for many years. The discovery of Z-DNA/RNA binding domains (Zα domains) in varied proteins that are involved in the innate immune response, such as the interferon inducible form of the RNA editing enzyme ADAR1 (p150), Z-DNA binding protein 1 (ZBP1), the fish kinase PKZ and the poxvirus inhibitor of interferon response E3L, indicates important roles of Z-DNA/RNA in immunity and self/non-self-discrimination. Such Zα domain-containing proteins recognize left-handed Z-DNA/RNA in a conformation-specific manner. Recent studies have implicated these domains in virus recognition. Given these important emerging roles for the Zα domains, it is pivotal to understand the mechanism of recognition of the Z-DNA/Z-RNA by these domains. To this end, we assessed the binding thermodynamics of Zα domain from ORF112 and ADAR1 on T(CG)3 and T(CG)6 oligonucleotides which have high propensity to adopt the Z-conformation. Our study highlights important differences in the mode of oligonucleotide binding by the two Zα domains originating from different proteins. Site-directed mutagenesis was employed together with isothermal titration calorimetry to tease apart finer details of the binding thermodynamics. Our work advances the understanding on binding thermodynamics of Zα domains to their cognate nucleic acid substrates and paves the ground for future efforts to gain a complete appreciation of this process.

HDAC3 Contributes to Ischemic Stroke by Regulating Interferon Pathway.

BACKGROUND: The inflammation and immune response contribute to ischemic stroke pathology. Damaged brain cells release inflammatory substances to activate the immune system in the acute phase of stroke, including altering the interferon signaling pathway. However, the involvement of histone deacetylation in stroke remains unclear. METHODS: To investigate whether histone deacetylation modulation could regulate the interferon signaling pathway and mediate the pathogenic changes after stroke, the middle cerebral artery occlusion (MCAO) mouse model was treated with histone deacetylase 3 (HDAC3) inhibitor and RGFP966. Additionally, a series of approaches, including middle cerebral artery occlusion (MCAO), real-time polymerase chain reaction (PCR), western blot, 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavioral experiments, and confocal imaging were utilized. RESULTS: It is observed that RGFP966 pretreatment could lead to better outcomes in the MCAO mouse model, including the decrease of infarction volumes, the amelioration of post-stroke anxiety-like behavior, and the relief of inflammatory responses. Furthermore, we found that RGFP966 could counteract the hyperactivation of the interferon signaling pathway and the excessive expression of Z-DNA Binding Protein 1 (ZBP1) in microglia. CONCLUSIONS: We demonstrated a novel mechanism that HDAC3 inhibition could ameliorate the pathological injury after ischemic stroke by downregulating the ZBP1/phosphorylated Interferon Regulatory Factor 3 (p-IRF3) pathway. Thus, these data provide a new promising target for therapies for ischemic stroke.

Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions.

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural-functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.