Phase separation in cGAS-STING signaling

Abstract

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

Keywords

biomolecular condensates / phase separation / cGAS-STING pathway / cGAS / STING / cGAMP / interferon

1 Introduction

Detection of various pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) by pattern recognition receptors (PRRs) is critical for the host to initiate innate immune responses. Unique among the many PRRs is the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), which recognizes aberrant DNA in the cytoplasm resulting from pathogen infection and cellular stress [1,2]. Upon binding to DNA, cGAS is activated to synthesize the second messenger cyclic GMP-AMP (cGAMP), which, in turn, activates stimulator of interferon genes (STING) at the endoplasmic reticulum [1–9]. Activated STING then translocates to the Golgi apparatus, where sulfated glycosaminoglycans further polymerize STING to recruit and activate TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) [10–14]. Subsequently, the activated IRF3 enters the nucleus to induce the expression of type I interferon (IFN-I) (Fig.1) [15–18]. Studies have shown that cGAS-STING signaling is involved in diverse cellular functions, including anti-infection defense, antitumor immunity, autophagy induction, cell senescence, protein synthesis, lipid and glucose metabolism, DNA damage repair, and cell death [19]. However, abnormal activation of cGAS-STING signaling is implicated in many diseases: loss-of-function mutations in cGAS-STING signaling lead to hypoimmunity and oncogenesis; gain-of-function mutations in cGAS-STING signaling and aberrant DNA accumulation lead to autoimmune and inflammatory diseases, such as Aicardi–Goutières syndrome and systemic lupus erythematosus [19–22]. Therefore, cGAS-STING signaling is of biomedical importance and has been considered as a potential therapeutic target for investigation. Over the past decade, research on drugs targeting cGAS-STING signaling has made good progress, including the development of agonists to activate antitumor immunity in immunosuppressed tumors and the development of antagonists to suppress the innate immune response in autoimmune and inflammatory diseases [22–25].

Biomolecular condensates, also known as membraneless organelles, form by phase separation driven by weak multivalent interactions involving multiple copies of structural motifs or intrinsic disorder regions (IDRs) of biomolecules [26–30]. This mechanism allows the partitioning and concentration of specific molecules in a microreactor when the concentration of biomolecules exceeds the saturation concentration. In some cases, phase separation of biomolecules undergoes a transition from liquid-like to gel-like or solid state, accompanied by a decrease in molecular dynamics. With these properties, phase separation can modulate the concentration, composition, and dynamics of molecules, thus playing a critical role in the regulation of diverse physiologic processes such as embryonic development, synaptic signaling, and cellular response to stress. In addition, biomolecular condensates have also been reported to be associated with various diseases, including cancer, neurodegeneration, and infectious diseases [31]. The emerging understanding of the functions and mechanisms of biomolecular condensates provides a novel angle to understand physiological processes and diseases, and also provides a new opportunity for drug discovery [32].

Phase separation has been characterized in immune responses, including T cell receptor signaling, B cell receptor signaling, and certain innate immune signaling pathways [33]. Recently, phase separation in cGAS-STING signaling involving cGAS condensates, STING condensates, and IRF3 condensates has been reported. These studies revealed novel activation and regulation mechanisms of cGAS-STING signaling and provided a new opportunity for drug discovery. Here, we briefly review the recent progress on phase separation in cGAS-STING signaling and the development of potential drugs targeting condensates.

2 cGAS condensates

As an innate immune DNA sensor, cGAS recognizes aberrant DNA through both the N-terminal disorder region and the C-terminal catalytic domain. Structural data show that the catalytic domain of human cGAS has three major DNA binding sites: site A, site B, and site C [6,34]. Site A and site B bind to DNA cooperatively and induce the formation of a 2:2 cGAS-DNA complex, which is required for cGAS to adopt the stable active conformation [4,6,35]. Site C has been identified as an additional cGAS-DNA interface and promotes the formation of higher-order cGAS-DNA complexes [34]. In addition, the positively charged N-terminal disorder region has also been shown to contribute to non-specific DNA binding and cGAS activation [36]. Recent works have shown that these multivalent interactions between the multiple DNA binding sites of cGAS and the phosphate backbone of DNA drive the DNA-induced liquid-liquid phase separation (LLPS) of cGAS (Fig.2) [34,37,38].

Multivalent elements involving DNA length and DNA binding sites of cGAS are necessary for cGAS-DNA phase separation. With higher valency, long DNA induces stronger phase separation than short DNA, consistent with long DNA activating cGAS more efficiently [37]. In contrast, deletion of the cGAS N terminus, mutation of the cGAS DNA binding site, and tumor-associated mutations within site C have been shown to decrease valency and thus weaken phase separation [34,37,39]. Compared to mouse cGAS, two loops (N389-C405 and E422-S434) within the catalytic domain of human cGAS contribute to multivalent interactions, thereby enhancing phase separation of human cGAS [39].

Further research has shown that DNA-induced LLPS of cGAS is critical for cGAS-mediated innate immune signaling. This condensation process allows cGAS, DNA, and substrates ATP/GTP to be rapidly concentrated into a microreactor, resulting in efficient detection of aberrant cytosolic DNA above a certain threshold and promotion of cGAMP production [37]. In addition, the condensation process has also been shown to promote cGAS activation by suppressing negative regulators. cGAS-DNA condensates can function as a selective filter that confines the DNA exonuclease activity of TREX1, a key negative regulator of cytosolic DNA sensing, to an outer shell at the periphery of the cGAS-DNA droplet, thereby protecting DNA from degradation by TREX1 (Fig.2) [20,39,40]. Another negative regulator, BAF, is also repressed by cGAS-DNA condensates in a manner similar to TREX1 [39]. Taken together, the DNA-induced LLPS of cGAS not only contributes to the rapid response to DNA and enhances cGAS activity, but also suppresses the inhibitory effects of negative regulators.

3 Host-mediated regulation of cGAS condensates

Given the importance of cGAS phase separation for innate immune signaling and immune homeostasis, host cells encode a variety of protein factors to regulate cGAS-DNA phase separation. Ku proteins (Ku70 and Ku80) have been identified as co-sensors of cGAS that directly interact with cGAS and enhance the DNA binding ability of cGAS to promote cGAS-DNA phase separation [41]. Similarly, Zyg-11 family member B (ZYG11B) has also been reported to be associated with cGAS and enhance the DNA binding ability to promote cGAS-DNA phase separation [42]. PCBP1 and PCBP2 both belong to the poly(C) binding protein (PCBP) family, which bind to poly(C) stretches of RNA and DNA [43]. PCBP1 has been reported to be associated with cGAS and to enhance cGAS binding to DNA after viral infection [44]. Researchers observed that PCBP1 colocalizes with cGAS-DNA condensates in cells, suggesting that it may be important for cGAS-DNA phase separation [44]. In contrast to PCBP1, PCBP2 has been proposed to act as a negative regulator by accumulating at the periphery and reducing the size of cGAS condensates [45]. Inflammasome activation has been shown to dampen cGAS-dependent signaling through caspase 1-mediated cleavage of the N terminus of cGAS [46,47]. Since the N-terminal disorder region is critical for cGAS phase separation, this cleavage process may restrict cGAS phase separation.

Several host proteins modulate cGAS condensates depending on their own phase separation ability. The deubiquitinase ubiquitin carboxyl-terminal hydrolase 15 (USP15) has been reported to promote cGAS activation [48]. USP15 not only mediates the deubiquitylation of cGAS, but also forms condensates with cGAS through its intrinsic negatively charged disordered region, which then promotes cGAS-DNA phase separation (Fig.2) [48]. GTPase-activating protein SH3 domain binding protein 1 (G3BP1) is a core member of stress granules (SGs) in response to cellular stress and has been shown to promote DNA binding and activation of cGAS by interacting with the N-terminal region of cGAS [49–51]. Further research has shown that G3BP1 engages cGAS in the primary condensation state, which then recruits DNA to rapidly form cGAS-DNA condensates and trigger the dissociation of G3BP1 (Fig.2) [52]. Other research has also observed that G3BP1-cGAS condensates contain protein kinase R (PKR) in cells, and proposed that both G3BP1 and PKR are required for DNA recognition of cGAS [53].

Other proteins have been identified as co-sensors of cGAS, including NONO (non-POU domain-containing octamer binding protein), PQBP1 (polyglutamine binding protein 1), IFI16 (interferon-γ inducible protein 16), and ZCCHC3 (CCHC-type zinc-finger protein 3) [54–59]. However, whether and how they are involved in cGAS condensates is not clear and remains to be explored.

In addition to protein factors, other cellular components, such as zinc ions, RNA, and certain metabolites, have also been reported to modulate cGAS condensates. Zinc ions have been shown to promote cGAS-DNA phase separation by stabilizing the 2:2 cGAS-DNA complex under physiologic conditions [37]. Although RNA cannot directly activate cGAS, it has been shown to induce cGAS to form condensates, providing a platform to promote the formation of cGAS-DNA condensates at low DNA concentrations (Fig.2) [37,60]. When the concentration of DNA is high enough to replace RNA in cGAS condensates, RNA has been shown to inhibit cGAS activation [60]. These data suggest that DNA and RNA in cGAS condensates are likely in a dynamic equilibrium to ensure the optimal activation of cGAS. Spermine, a host-synthesized polyamine, has been shown to condense DNA in a charge-dependent manner and is used by viruses to condense their DNA into virions [61–70]. Recent research has shown that spermine promotes cGAS-DNA phase separation by condensing DNA to a similar inter-DNA distance in the cGAS-DNA complex (Fig.2) [70]. Thus, spermine has been shown to play a critical role in cGAS-mediated antiviral and antitumor effects [70]. It has been suggested that spermine contributes to the self/non-self discrimination of cGAS, as it condenses naked DNA but not nucleosomal DNA [70]. In contrast, another cellular metabolite, oleic acid, has been shown to dissolve cGAS-DNA phase separation to attenuate cGAS-STING signaling [71].

4 Pathogen-mediated regulation of cGAS condensates

It is not surprising that pathogens have evolved multiple regulatory strategies, including the restriction of cGAS-DNA phase separation, to evade cGAS-STING immune responses. ORF52/VP22-type viral proteins, an evolutionarily divergent but structurally related family of tegument proteins from gamma- and alpha-herpesvirinae, have been identified as antagonists of cGAS to suppress the innate immune response [72–74]. With an intrinsically disordered region and a core domain containing multivalent DNA-interacting surfaces, ORF52/VP22-type tegument proteins have been shown to undergo LLPS to form condensates with DNA (Fig.2) [75,76]. Depending on the stronger phase separation ability with DNA rather than higher DNA binding affinity or strong direct protein–protein interaction, ORF52/VP22-type tegument proteins disrupt cGAS-DNA phase separation to form their own condensates with DNA, thereby inhibiting cGAS activation [75,76]. As a positive-sense RNA virus, SARS-CoV-2 has been shown to induce mitochondrial DNA (mtDNA) release and trigger cGAS-mediated immune responses after infection [77,78]. Interestingly, SARS-CoV-2 nucleocapsid protein (SARS2-NP) was reported to undergo DNA-induced LLPS to form N-DNA-G3BP1 condensates, thereby restricting the formation of cGAS-G3BP1 condensates and suppressing cGAS-mediated anti-SARS-CoV-2 immune responses (Fig.2) [78]. On the other hand, streptavidin, a secreted protein from the soil bacterium Streptomyces avidinii and a widely used biological and clinical tool, has been reported to promote cGAS activation by directly binding to cGAS and enhancing DNA binding ability to promote cGAS-DNA phase separation [79]. These findings highlight the critical role of phase separation not only in host-mediated anti-infection defense, but also in pathogen-mediated immune evasion.

5 STING condensates

STING is an ER-localized dimeric protein containing a N-terminal transmembrane domain, a cytosolic ligand binding domain (LBD), and a downstream factor-recruiting C-terminal tail (CTT). Structural data show that apo-STING forms bilayer oligomers through the LBD, which has been proposed to be critical for STING ER retention and autoinhibition [80]. Upon binding to cGAMP, apo-STING undergoes significant conformational changes, represented by an open-to-closed transition of the LBD and a ~180° rotation between the LBD and the transmembrane domain, thereby resulting in the formation of STING filament [5,25,81]. The activated STING filament adopts a bent conformation that is thought to be suitable for translocation via COP-II vesicles to the Golgi apparatus, where STING is further polymerized by sulfated glycosaminoglycans [13,80,82]. High-order oligomerization of STING serves as a platform for recruitment and activation of TBK1 and IRF3 [83–85].

However, continuous activation of cGAS produces excess cGAMP. Recent research has shown that excessive cGAMP induces the formation of STING condensates, also known as STING phase-separator, which functions to suppress innate immune signaling (Fig.2) [86]. In a fluid-to-gel transition, STING condensates act as a “STING-TBK1-cGAMP sponge” to recruit TBK1 and separate STING and TBK1 from IRF3, thereby suppressing signal transduction and preventing STING from overactivation [86]. The STING membrane topology, the IDR segment, and the dimerization region are all required for the formation of STING condensates [86]. Other CDNs, such as c-di-GMP, have been shown to require higher concentrations to induce the formation of STING condensates [86]. In addition, Mn²⁺ is released from mitochondria and the Golgi apparatus upon viral infection and has been shown to promote STING phase separation and the gel-like transition by enhancing the cGAMP binding ability of STING [86,87].

6 IRF3 condensates

From the N- to C-terminus, IRF3 is composed of a DNA binding domain (DBD), an intrinsic disorder region (IDR), an IRF-associated domain (IAD), and an inhibitory domain (ID). Phosphorylation of the C-terminus induces IRF3 to release autoinhibition and form a dimer through the IAD. Activated IRF3 allows the N-terminal DBD to bind to IFN-stimulated response elements (ISREs) in IFN-I promoters, which then induces IFN-I expression [88–90]. Recent research has reported that IRF3 undergoes LLPS with ISREs DNA in the nucleus, which requires the IDR, DBD and IAD regions (Fig.2) [91]. As a homolog and associated protein of IRF3, IRF7 has also been shown to undergo LLPS and be compartmentalized by IRF3 condensates [91]. Interestingly, the super-enhancer mark mediator of RNA polymerase II transcription subunit 1 (MED1) is enriched in IRF3 condensates, whereas the transcriptional repressor mark H3K9me3 is segregated from IRF3 condensates [91]. It is proposed that IRF3 condensates function to recruit transcription factors and co-activators, thereby promoting IFN-I expression [91].

7 Regulation of IRF3 condensates

Further research has shown that IRF3 phase separation is regulated by the deacetylase activity of SIRT1 [91]. As a NAD⁺-dependent deacetylase, SIRT1 has been proposed to mediate the deacetylation of IRF3. In the absence of SIRT1, IRF3 was found to be hyperacetylated at two conserved lysine residues in the DBD, resulting in the destruction of its ability to bind ISREs and phase separation [91]. Thus, the deacetylase activity of SIRT1 appears to be critical for IRF3 phase separation. Due to the degradation of SIRT1 and the decrease in NAD⁺ levels with age, the deacetylase activity of SIRT1 is lower in aging individuals than in young individuals [92,93]. The age-related decline in SIRT1 activity then impairs the innate immune response mediated by IRF3 phase separation, leading to innate immunosenescence [91]. This may partially explain the much higher rates of hospitalization and death in aging individuals during influenza and COVID-19 pandemics [91,94,95].

8 Phase separation associated diseases in cGAS-STING signaling

A growing body of research has shown that aberrant forms of condensates are associated with many human diseases, including cancer, neurodegeneration, and infectious diseases [31]. Given that phase separation is important for cGAS-STING signaling, loss-of-function mutations in cGAS-STING pathway that weaken phase separation may impair pathogen defense and antitumor responses mediated by cGAS-STING signaling. As previously discussed, two tumor-associated mutations, G303E and K432T, within the DNA binding site C of cGAS have been shown to weaken cGAS-DNA phase separation and have been proposed to be responsible for oncogenesis [34]. In contrast, gain-of-function mutations in STING and loss-of-function mutations in negative regulators of cGAS-STING signaling can cause autoimmune diseases, including systemic lupus erythematosus (SLE), familial chilblain lupus erythematosus (FCL), Aicardi–Goutières syndrome (AGS), STING-associated vasculopathy with onset in infancy (SAVI), and type I interferonopathy [22]. STING mutations within the F153-P173 region, such as V147L, N154S or V155M, have been reported to be associated with autoimmune diseases [96–98]. Further research has shown that these STING mutations reduce STING condensates, leading to overactivation of STING [86]. These data provide a link between aberrant cGAS-STING condensates and different diseases.

In addition, disease-associated mutations in other regulators have been proposed to induce aberrant condensates and limit cGAS-STING signaling. Neurofibromin 2 (NF2) is a tumor suppressor that contributes to enhanced nucleic acid sensing by relieving YAP/TAZ-mediated TBK1 inhibition [99]. However, patient-derived individual mutations in the FERM domain of NF2 (NF2m) have been shown to suppress nucleic acid sensing and subsequently lead to the development of neurofibromatosis type 2 and the occurrence of multiple malignancies [100,101]. Recent research has shown that mutations within the FERM domain cause NF2m to undergo a conformational change, allowing NF2m to interact with activated IRF3 to form liquid condensates (Fig.2) [99]. NF2m condensates act as a platform for the recruitment of TBK1, numerous protein kinases, protein phosphatases, and ubiquitin E3 ligases [99]. As a phosphatase, the RACK1-PP2A complex is also recruited to NF2m condensates, where the RACK1-PP2A complex dephosphorylates TBK1 to prevent its activation, thereby suppressing innate immune signaling [99].

9 Drugs targeting condensates

Since condensates are widely distributed in normal physiologic processes and diseases, the design of drugs targeting condensates would be a potential therapeutic strategy. Recently, it has been discussed that the drugs targeting condensates should be designed to modulate the physical properties, macromolecular network, composition, dynamics, and function of specific biomolecular condensates, thereby achieving goals such as repairing a condensate that drives a specific disease phenotype, disrupting the functions of condensates associated with disease, delocalizing a specific target from its native condensate, or rendering it inactive within the condensate [32].

Several studies have made great progress in the development of potential drugs that target condensates. Human respiratory syncytial virus (RSV) replication relies on the formation of inclusion bodies (IBs), condensates that concentrate the transcription factor M2-1, viral genomic RNA, and other factors [102–105]. Identified as a potent inhibitor of RSV replication, the Hedgehog (HH) pathway antagonist cyclopamine (CPM) and its chemical analog A3E were shown to disorganize and harden IB condensates, thereby inhibiting RSV replication [106,107]. SARS-CoV-2 nucleocapsid protein (SARS2-NP) has been reported to undergo LLPS with RNA to suppress the antiviral immune response mediated by MAVS (mitochondrial antiviral-signaling protein) [108]. Since LLPS of SARS2-NP relies on the dimerization domain, researchers designed a peptide that interferes with the interaction between the dimerization domain of SARS2-NP [108]. Treatment with this peptide was shown to disrupt the LLPS of SARS2-NP, thereby promoting the MAVS-mediated antiviral immune response [108]. These studies are representative examples of achieving antiviral therapy by disrupting the critical condensates of the virus, and suggest that condensates are a potential target for drug development.

10 Drugs targeting condensates of cGAS-STING signaling

Progress has also been made in the development of potential drugs that target condensates of the cGAS-STING pathway. As noted above, G3BP1 promotes cGAS activation through interacting with cGAS to form cGAS-G3BP1 condensates [52]. Epigallocatechin gallate (EGCG), a natural G3BP1 inhibitor isolated from green tea leaves, was found to be able to suppress cGAS activation by inhibiting the formation of G3BP1-promoted cGAS condensates [51,52,109]. Thus, EGCG has been suggested as a potential drug for cGAS-related autoimmune diseases [51].

Given that SIRT1 activity is critical for the formation of IRF3 condensates, enhancing SIRT1 activity to promote IRF3 phase separation may contribute to elevate the innate immune response in aging individuals with innate immunosenescence [91]. Identified as SIRT1 agonists, the natural compound resveratrol (SRT501) and the chemically synthetic compound SRT2183 have been shown to rescue IRF3 phase separation by enhancing the deacetylase activity of SIRT1, thereby promoting innate immune responses mediated by IRF3 phase separation and thus preventing innate immunosenescence in aging individuals [91,110,111].

Since multivalent interactions are critical for phase separation, the design of polyvalent agonists to induce phase separation of cGAS-STING signaling would be an effective immunotherapy. PC7A, a pH-sensitive polymer bearing a seven-membered ring with a tertiary amine, was synthesized and served as a polyvalent STING agonist to activate antitumor immunity for cancer immunotherapy [112]. Compared with high concentration of cGAMP, which induces the formation of gel-like STING condensates to suppress innate immune signaling, PC7A binds to the negatively charged surface (E296-D297) of STING and induces the formation of liquid-like STING-PC7A condensates to activate innate immune signaling (Fig.2) [113]. As a polymer with multivalent properties, PC7A induces the formation of STING-PC7A condensates through multivalent interactions. PC7A with a higher degree of polymerization has more valence, leading to stronger phase separation [113]. However, too long a PC7A chain (> 70 repeating units) induces STING to undergo a liquid-to-gel transition, thereby suppressing STING activation [113]. Although PC7A induces a sustained activation of STING, cGAMP-induced STING activation is faster than that of PC7A [113]. Combining the advantages of cGAMP and PC7A, cGAMP-PC7A nanoparticles were designed to achieve rapid and sustained STING activation [113].

11 Conclusions

Biomolecular condensates are widespread in cGAS-STING signaling and play a critical role in the activation and regulation of the innate immune response. First, aberrant DNA from pathogens or the host induces cGAS to form cGAS-DNA condensates, which contribute to promote cGAS activation and suppress the inhibitory effects of negative regulators. Appropriate levels of cGAMP then induce STING to translocate from ER to Golgi apparatus, where STING activates TBK1 and IRF3, while excessive cGAMP induces the formation of the STING phase separator to prevent STING from overactivation. Subsequently, activated IRF3 forms condensates with ISREs to stimulate IFN-I expression. Meanwhile, positive or negative regulators from the host modulate phase separation processes of cGAS-STING signaling to maintain immune homeostasis in various ways, such as phase separation, direct interaction, and post-transcriptional modifications (Tab.1). Pathogens such as herpesviruses and SARS-CoV-2 also employ phase separation to restrict the formation of cGAS-DNA condensates to achieve immune evasion (Tab.1). Importantly, several mutations in cGAS-STING signaling cause aberrant condensates that are responsible for the development of associated diseases, including hypoimmunity, oncogenesis, autoimmune and inflammatory diseases. Antagonists and agonists that modulate the phase separation of cGAS-STING signaling have been identified, developed, and shown to have therapeutic potential.

Research on the biomolecular condensates provides a novel angle to understand the activation and regulation mechanisms of cGAS-STING signaling. Although previous structural and functional studies have revealed the mechanisms of direct and strong interactions between structured domains, the functions of weak multivalent interactions and unstructured regions in cGAS-STING signaling have been overlooked. Phase separation not only highlights the importance of these interactions and regions in cGAS-STING signaling, but also provides potential new therapeutic targets for cancer and autoimmune diseases.

Despite these exciting advances, there are still several open questions regarding the condensates of cGAS-STING signaling. Although some of the regulators involved in the regulation of the condensates in cGAS-STING signaling have been identified, the detailed composition and organization of condensates in the complex cellular environment is still not clear. As a common regulatory mechanism, post-transcriptional modifications have been reported to play critical roles in regulating biomolecular condensates [114,115]. However, the mechanisms by which post-transcriptional modifications regulate cGAS condensates and STING condensates are poorly understood. With regard to the development of drugs targeting condensates, the efficacy and safety of the drug should be seriously considered. These questions remain to be addressed in the future.

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