Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.     

Regulation of cGAS-STING signalling in cancer: Approach for combination therapy

Innate immunity plays an important role not only during infection but also homeostatic role during stress conditions. Activation of the immune system including innate immune response plays a critical role in the initiation and progression of tumorigenesis. The innate immune sensor recognizes pathogen-associated molecular patterns (PAMPs) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (cGAS-STING) and induces type-1 immune response during viral and bacterial infection. cGAS-STING is regulated differently in conditions like cellular senescence and DNA damage in normal and tumor cells and is implicated in the progression of tumors from different origins. cGAS binds to cytoplasmic dsDNA and synthesize cyclic GMP-AMP (2’3’-cGAMP), which selectively activates STING and downstream IFN and NF-κB activation. We here reviewed the cGAS-STING signalling pathway and its cross-talk with other pathways to modulate tumorigenesis. Further, the review also focused on emerging studies that targeted the cGAS-STING pathway for developing targeted therapeutics and combinatorial regimens for cancer of different origins.     

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.     

Single Amino Acid Change in STING Leads to Constitutive Active Signaling

The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS) activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.     

RNF111-facilitated neddylation potentiates cGAS-mediated antiviral innate immune response

The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) has emerged as a fundamental component fueling the anti-pathogen immunity. Because of its pivotal role in initiating innate immune response, the activity of cGAS must be tightly fine-tuned to maintain immune homeostasis in antiviral response. Here, we reported that neddylation modification was indispensable for appropriate cGAS-STING signaling activation. Blocking neddylation pathway using neddylation inhibitor MLN4924 substantially impaired the induction of type I interferon and proinflammatory cytokines, which was selectively dependent on Nedd8 E2 enzyme Ube2m. We further found that deficiency of the Nedd8 E3 ligase Rnf111 greatly attenuated DNA-triggered cGAS activation while not affecting cGAMP induced activation of STING, demonstrating that Rnf111 was the Nedd8 E3 ligase of cGAS. By performing mass spectrometry, we identified Lys231 and Lys421 as essential neddylation sites in human cGAS. Mechanistically, Rnf111 interacted with and polyneddylated cGAS, which in turn promoted its dimerization and enhanced the DNA-binding ability, leading to proper cGAS-STING pathway activation. In the same line, the Ube2m or Rnf111 deficiency mice exhibited severe defects in innate immune response and were susceptible to HSV-1 infection. Collectively, our study uncovered a vital role of the Ube2m-Rnf111 neddylation axis in promoting the activity of the cGAS-STING pathway and highlighted the importance of neddylation modification in antiviral defense.     

Cancer cell-specific cGAS/STING Signaling pathway in the era of advancing cancer cell biology

Pattern-recognition receptors (PRRs) are critical to recognizing endogenous and exogenous threats to mount a protective proinflammatory innate immune response. PRRs may be located on the outer cell membrane, cytosol, and nucleus. The cGAS/STING signaling pathway is a cytosolic PRR system. Notably, cGAS is also present in the nucleus. The cGAS-mediated recognition of cytosolic dsDNA and its cleavage into cGAMP activates STING. Furthermore, STING activation through its downstream signaling triggers different interferon-stimulating genes (ISGs), initiating the release of type 1 interferons (IFNs) and NF-κB-mediated release of proinflammatory cytokines and molecules. Activating cGAS/STING generates type 1 IFN, which may prevent cellular transformation and cancer development, growth, and metastasis. The current article delineates the impact of the cancer cell-specific cGAS/STING signaling pathway alteration in tumors and its impact on tumor growth and metastasis. This article further discusses different approaches to specifically target cGAS/STING signaling in cancer cells to inhibit tumor growth and metastasis in conjunction with existing anticancer therapies.     

KSHV strategies for host dsDNA sensing machinery

The innate immune system utilizes pattern recognition receptors cyclic GMP-AMP synthase(cGAS)to sense cytosolic double-stranded(ds) DNA and initiate type 1 interferon signaling and autophagy pathway, which collaborate to limit pathogen infections as well as alarm the adaptive immune response. The genomes of herpesviruses are large dsDNA, which represent a major class of pathogen signatures recognized by cellular DNA sensor cGAS. However, to successfully establish the persistent infection, herpesviruses have evolved their viral genes to modulate different aspects of host immune signaling. This review summarizes the evasion strategies of host cGAS DNA sensing pathway by Kaposi's Sarcoma-associated Herpesvirus(KSHV) and their contributions to KSHV life cycles.     

cGAS is activated by DNA in a length‐dependent manner

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP‐AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length‐dependent manner. This is observed over a wide length‐span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length‐dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.     

Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62‐deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy‐associated vesicles. Thus, DNA sensing induces the cGAS‐STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.     

Autophagy side of MB21D1/cGAS DNA sensor

The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.     

Recognition of Cytosolic DNA Attenuates Glucose Metabolism and Induces AMPK Mediated Energy Stress Response

Both viral infection and DNA transfection expose single-stranded or double-stranded DNA to the cytoplasm of mammalian cells. Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway. Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types. Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1. Our results suggest that in concert with but independent of innate immune response, recognition of cytosolic DNA induced cellular energy stress potentially functions as a metabolic barrier to viral replication.     

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.     

cGAS exacerbates Schistosoma japonicum infection in a STING-type I IFN-dependent and independent manner

Schistosomiasis, which is caused by infection with Schistosoma spp., is characterized by granuloma and fibrosis in response to egg deposition. Pattern recognition receptors are important to sense invading Schistosoma, triggering an innate immune response, and subsequently shaping adaptive immunity. Cyclic GMP-AMP synthase (cGAS) was identified as a major cytosolic DNA sensor, which catalyzes the formation of cyclic GMP-AMP (cGAMP), a critical second messenger for the activation of the adaptor protein stimulator of interferon genes (STING). The engagement of STING by cGAMP leads to the activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and the subsequent type I interferon (IFN) response. cGAS is suggested to regulate infectious diseases, autoimmune diseases, and cancer. However, the function of cGAS in helminth infection is unclear. In this study, we found that Cgas deficiency enhanced the survival of mice infected with S. japonicum markedly, without affecting the egg load in the liver. Consistently, Cgas deletion alleviated liver pathological impairment, reduced egg granuloma formation, and decreased fibrosis severity. In contrast, Sting deletion reduced the formation of egg granulomas markedly, but not liver fibrosis. Notably, Cgas or Sting deficiency reduced the production of IFNβ drastically in mice infected with S. japonicum. Intriguingly, intravenous administration of recombinant IFNβ exacerbated liver damage and promoted egg granuloma formation, without affecting liver fibrosis. Clodronate liposome-mediated depletion of macrophages indicated that macrophages are the major type of cells contributing to the induction of the type I IFN response during schistosome infection. Moreover, cGAS is important for type I IFN production and phosphorylation of TBK1 and IRF3 in response to stimulation with S. japonicum egg- or adult worm-derived DNA in macrophages. Our results clarified the immunomodulatory effect of cGAS in the regulation of liver granuloma formation during S. japonicum infection, involving sensing schistosome-derived DNA and producing type I IFN. Additionally, we showed that cGAS regulates liver fibrosis in a STING-type I–IFN-independent manner.     

Autophagy side of MB21D1/cGAS DNA sensor

The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.