GO:0106177 cyclic-GMP-AMP hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106177 describes the enzymatic activity that hydrolyzes cyclic GMP-AMP (cGAMP) into AMP and GMP, thereby terminating the cGAS-STING second messenger signal [2, 3].
• This hydrolase activity is a key negative regulator of cytosolic DNA sensing, preventing excessive type I interferon and NF-kB responses [1, 4, 5].
• Dysregulation of cGAMP hydrolysis is linked to autoinflammation, cancer immune evasion, and radiotherapy resistance [4, 6, 7].
• The cGAS-STING pathway, which produces cGAMP, is activated by cytosolic DNA from pathogens, damaged nuclei, or R-loop-derived RNA-DNA hybrids [1, 3, 8].
• Studying GO:0106177 requires precise measurement of cGAMP levels and downstream signaling, using methods such as LC-MS, reporter assays, and CRISPR knockout models [2, 7].
• EDITGENE provides CRISPR knockout, point-mutation, knock-in, overexpression, and library screening services to dissect cGAMP hydrolase function in disease models [2, 7].
Description
Cyclic GMP-AMP (cGAMP) is a second messenger produced by cGAS upon binding to cytosolic double-stranded DNA, and it activates STING to induce type I interferon and inflammatory cytokines [2, 3]. The enzymatic activity defined by GO:0106177, cyclic-GMP-AMP hydrolase activity, catalyzes the hydrolysis of cGAMP to AMP and GMP, thereby shutting off this signaling axis [2, 4]. This activity is essential for maintaining immune homeostasis and preventing autoinflammation, as excessive cGAS-STING signaling contributes to senescence, cancer, and tissue damage [3, 5, 6]. Researchers study GO:0106177 to understand how cells balance immune activation and tolerance, and to identify therapeutic targets for diseases ranging from autoimmunity to cancer [4, 7]. The hydrolase activity is mediated by multiple enzymes, including ENPP1 and other phosphodiesterases, and its regulation is critical for the duration and intensity of the cGAS-STING response [2, 7].
cyclic-GMP-AMP hydrolase activity At A Glance
| GO ID | GO:0106177 |
|---|---|
| GO term | cyclic-GMP-AMP hydrolase activity |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Hydrolysis of cyclic GMP-AMP to AMP and GMP, terminating cGAS-STING signaling [2, 4] |
| Reaction | cyclic GMP-AMP + 2 H2O = AMP + GMP |
| Related pathway | cGAS-STING cytosolic DNA sensing [2, 3] |
| Key regulators | ENPP1, TREX1, and other phosphodiesterases [4, 7] |
| Disease relevance | Autoinflammation, cancer, radiotherapy response [4, 6, 7] |
What Is GO:0106177?
GO:0106177, cyclic-GMP-AMP hydrolase activity, is a molecular function defined as the catalysis of the reaction: cyclic GMP-AMP + 2 H2O = AMP + GMP. In other words, it is the enzymatic breakdown of the cyclic dinucleotide cGAMP into its linear monophosphate components, AMP and GMP. This activity directly opposes the synthesis of cGAMP by cGAS and serves as a negative feedback mechanism in cytosolic DNA sensing pathways [2, 4].
Why Is cyclic-GMP-AMP hydrolase activity Important in Cell Biology?
GO:0106177 is critical because it controls the intensity and duration of the cGAS-STING immune response, which is central to host defense against pathogens and to cancer immunosurveillance [2, 5]. Without proper cGAMP hydrolysis, persistent STING activation can lead to chronic inflammation, autoimmune diseases, and tissue damage [3, 6]. Conversely, insufficient hydrolysis may enhance anti-tumor immunity but also risk autoimmunity [4, 7]. Understanding this activity provides opportunities for therapeutic modulation in oncology, infectious diseases, and inflammatory disorders [5, 8].
• Terminates cGAS-STING signaling to prevent excessive type I interferon production [2, 4].
• Regulates immune responses to cytosolic DNA from pathogens and damaged self-DNA [1, 3].
• Modulates radiotherapy-induced tumor immunogenicity by controlling cGAMP levels [4, 6].
• Influences senescence and aging through cytoplasmic chromatin-driven inflammation.
• Plays a role in platelet activation and thrombosis via cGAS-STING.
• Impacts cancer immune evasion and response to immune checkpoint blockade.
• Potential target for anti-inflammatory and anti-cancer therapies [5, 7].
• Involved in influenza spillover barrier through STING-NF-kB signaling.
• Key to understanding autoimmune diseases linked to DNA sensing [3, 6].
• Provides a mechanism for fine-tuning innate immune activation [2, 7].
Molecular Mechanism of cyclic-GMP-AMP hydrolase activity
Substrate recognition and binding
In simple terms: The enzyme grabs cGAMP and holds it in place to break it apart.
Cyclic GMP-AMP hydrolase enzymes, such as ENPP1, recognize the cyclic dinucleotide cGAMP through specific binding pockets that accommodate the two purine rings and the cyclic phosphate linkage [2, 7]. This binding is essential for the subsequent hydrolysis reaction, and structural studies have revealed key residues that coordinate the substrate.
Catalytic hydrolysis of cGAMP
In simple terms: Water molecules are used to split cGAMP into AMP and GMP.
The hydrolysis of cGAMP proceeds via a two-step mechanism in which two water molecules attack the phosphodiester bonds, resulting in the linear products AMP and GMP [2, 4]. This reaction is energetically favorable and effectively removes the cyclic dinucleotide that would otherwise activate STING.
Termination of STING signaling
In simple terms: By destroying cGAMP, the enzyme stops the immune alarm.
Once cGAMP is hydrolyzed, STING activation ceases, leading to downregulation of type I interferon and inflammatory cytokine production [2, 4]. This negative feedback is crucial for preventing sustained immune activation that could damage tissues [3, 6].
Regulation by cellular context
In simple terms: The enzyme's activity can be turned up or down depending on the cell's situation.
The expression and activity of cGAMP hydrolases are regulated by inflammatory cues, DNA damage, and cellular stress [4, 7]. For example, TREX1 degrades cytosolic DNA to limit cGAMP production, indirectly affecting hydrolase activity. Additionally, ubiquitination-directed cytosolic DNA degradation governs cGAS-STING-mediated immune responses, highlighting layers of regulation.
Key Genes Involved in GO:0106177 cyclic-GMP-AMP hydrolase activity
The following genes and proteins are directly or indirectly involved in cyclic-GMP-AMP hydrolase activity and its regulatory network.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ENPP1 | Ectonucleotide pyrophosphatase/phosphodiesterase 1; hydrolyzes cGAMP to AMP and GMP | Key enzyme for cGAMP degradation; target for cancer and inflammation [2, 7] |
| TREX1 | DNA exonuclease that degrades cytosolic DNA, limiting cGAMP production | Regulates cGAS-STING activation; mutations cause autoimmunity |
| CGAS | Synthesizes cGAMP from ATP and GTP upon DNA binding | Upstream of hydrolase activity; central to DNA sensing [2, 3] |
| STING1 | Adaptor protein activated by cGAMP; induces interferon and NF-kB | Downstream effector; target of cGAMP hydrolysis [2, 5] |
| NFKB1 | Transcription factor mediating inflammatory responses downstream of STING | Readout of cGAS-STING pathway activity |
| IRF3 | Transcription factor inducing type I interferon upon STING activation | Marker of cGAMP signaling [2, 3] |
| IFNB1 | Type I interferon gene induced by STING | Functional readout of cGAMP hydrolase activity [2, 4] |
| IL6 | Pro-inflammatory cytokine induced by STING-NF-kB | Inflammation marker |
| TNFA | Tumor necrosis factor alpha, induced by STING | Inflammation marker |
| CXCL10 | Chemokine induced by interferon signaling | Immune cell recruitment readout |
| ATG5 | Autophagy protein involved in STING trafficking | Links cGAMP signaling to autophagy |
| MAP1LC3B | Autophagy marker; STING trafficking induces autophagy | Readout of STING degradation |
| H2AX | DNA damage marker; cytoplasmic chromatin activates cGAS | Links DNA damage to cGAMP production |
| RAD51 | DNA repair protein; R-loop processing affects cytosolic DNA | Modulates cGAS activation |
| RNASEH1 | Ribonuclease that removes R-loops, preventing cytosolic RNA-DNA hybrids | Regulates cGAS activation |
| DDX41 | Cytosolic DNA sensor that can activate cGAS-STING | Alternative DNA sensing pathway |
| UBB | Ubiquitin; ubiquitination directs cytosolic DNA degradation | Regulates cGAS-STING |
| P2RX7 | Purinoceptor involved in platelet activation and cGAS-STING | Links cGAMP to thrombosis |
How Is cyclic-GMP-AMP hydrolase activity Regulated?
Cyclic-GMP-AMP hydrolase activity is regulated at multiple levels. The expression of enzymes such as ENPP1 is induced by inflammatory stimuli and DNA damage [4, 7]. Post-translational modifications, including ubiquitination, control the stability and localization of these enzymes. Additionally, the availability of substrate cGAMP is determined by the balance between cGAS synthesis and TREX1-mediated DNA degradation. Cellular stress, senescence, and radiotherapy can increase cytosolic DNA, thereby elevating cGAMP levels and engaging hydrolase activity as a feedback mechanism [3, 6]. Autophagy induction via STING trafficking also modulates the pathway by degrading STING, indirectly affecting cGAMP turnover.
cyclic-GMP-AMP hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ENPP1 | Cancer immune evasion, autoinflammation | ENPP1 knockout tumor cells; syngeneic mouse models [2, 7] |
| TREX1 | Radiotherapy resistance, autoimmunity | TREX1 knockout cells; radiation response assays |
| CGAS | Senescence, autoinflammation | cGAS knockout mice; senescence models |
| STING1 | Influenza, autoinflammatory syndromes | STING knockout mice; viral infection models |
| P2RX7 | Thrombosis, myocardial injury | Platelet-specific knockout; thrombosis models |
Cancer and radiotherapy response
In cancer, cGAMP hydrolase activity can limit anti-tumor immunity by reducing STING activation. Radiotherapy induces DNA damage and cytosolic DNA, leading to cGAMP production; however, TREX1 upregulation degrades this DNA and blunts the immune response [4, 6]. Inhibiting cGAMP hydrolysis may enhance radiotherapy-induced tumor immunogenicity and improve checkpoint blockade efficacy [6, 7].
Autoinflammatory and autoimmune diseases
Deficiency in cGAMP hydrolysis or upstream DNA degradation leads to chronic STING activation, causing autoinflammatory conditions such as Aicardi-Goutieres syndrome [3, 4]. Cytoplasmic chromatin in senescence also triggers inflammation, contributing to age-related pathologies.
Cardiovascular disease and thrombosis
Double-stranded DNA enhances platelet activation and thrombosis via cGAS, and cGAMP hydrolase activity may modulate this process. Dysregulated cGAMP signaling in platelets contributes to myocardial injury, suggesting a role for hydrolase activity in cardiovascular disease.
Infectious disease and inflammation
STING-NF-kB signaling builds an influenza spillover barrier, and cGAMP hydrolysis may influence the intensity of this response. Inflammatory microenvironment remodeling by tumor cells after radiotherapy also involves cGAMP dynamics.
From cyclic-GMP-AMP hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ENPP1 knockout enhance cGAMP-mediated STING activation? | ENPP1 knockout cell lines (e.g., HeLa, THP-1) [2, 7] |
| How does TREX1 mutation affect radiotherapy-induced immunity? | TREX1 point-mutation knock-in mice |
| Can cGAMP hydrolase activity be monitored in live cells? | STING reporter knock-in cells |
| What is the role of cGAMP hydrolysis in platelet activation? | Platelet-specific ENPP1 overexpression |
| Does cGAMP hydrolase regulate senescence-associated inflammation? | cGAS/STING knockout senescence models |
| Can CRISPR library screening identify novel cGAMP hydrolases? | Genome-wide CRISPR knockout library in reporter cells |
How to Study the cyclic-GMP-AMP hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| LC-MS | cGAMP, AMP, GMP levels | Direct measurement of hydrolase activity [2, 7] |
| IFN-beta luciferase reporter | STING-dependent transcription | Screening for modulators [2, 4] |
| CRISPR knockout screen | Gene requirements for cGAMP response | Discovery of novel regulators |
| Western blot | STING, phospho-TBK1, IRF3 | Pathway activation status [2, 5] |
| qRT-PCR | IFNB1, IL6, CXCL10 mRNA | Inflammatory gene expression |
| Immunofluorescence | STING localization | Trafficking and autophagy |
| Flow cytometry | Platelet activation markers | Thrombosis studies |
| ELISA | Cytokine secretion | Functional immune readout |
Quantification of cGAMP levels
Liquid chromatography-mass spectrometry (LC-MS) is used to measure cGAMP and its hydrolysis products AMP and GMP in cell extracts [2, 7]. This method provides direct evidence of hydrolase activity and is essential for validating enzyme function.
Reporter assays for STING activation
STING-dependent reporter cell lines (e.g., IFN-beta luciferase) are used to assess downstream signaling after cGAMP hydrolysis [2, 4]. These assays are high-throughput and suitable for screening modulators of hydrolase activity.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes that regulate cGAMP levels and STING activation. This approach has revealed novel components of the cGAS-STING pathway and potential hydrolases.
Imaging of STING trafficking
Fluorescence microscopy of tagged STING allows visualization of its trafficking to autophagosomes, which is linked to cGAMP signaling. This method provides spatial and temporal information on pathway regulation.
How CRISPR Can Be Used to Study GO:0106177 cyclic-GMP-AMP hydrolase activity
Knockout
CRISPR knockout of ENPP1 or other candidate hydrolases can confirm their role in cGAMP degradation. Knockout cells show elevated cGAMP levels and enhanced STING activation upon DNA stimulation [2, 7]. This approach is fundamental for establishing causality.
Point Mutation
Point mutations in the catalytic domain of hydrolases can abolish enzymatic activity while preserving protein structure. Such models are useful for dissecting the specific contribution of hydrolase activity versus other functions.
Knock-in
Knock-in of tagged hydrolases (e.g., HA-ENPP1) allows for localization and interaction studies. Additionally, knock-in of disease-associated mutations (e.g., TREX1) can model autoinflammatory syndromes.
Overexpression
Overexpression of cGAMP hydrolases reduces STING signaling and can suppress immune responses. This is useful for testing whether increased hydrolysis protects against autoinflammation or limits anti-tumor immunity [2, 7].
How EDITGENE Supports cyclic-GMP-AMP hydrolase activity Research
Researchers studying cyclic-GMP-AMP hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in cGAMP turnover and STING signaling. EDITGENE provides a comprehensive suite of CRISPR services to enable precise genetic manipulation and functional validation.
Contact EDITGENE today to design your custom CRISPR model for cyclic-GMP-AMP hydrolase activity research.
Frequently Asked Questions About cyclic-GMP-AMP hydrolase activity
What is cyclic-GMP-AMP hydrolase activity?
It is the enzymatic activity that breaks down cyclic GMP-AMP (cGAMP) into AMP and GMP, as defined by GO:0106177 [2, 4].
What genes are involved in cyclic-GMP-AMP hydrolase activity?
Key genes include ENPP1, TREX1, and other phosphodiesterases that regulate cGAMP levels [2, 4, 7].
How does cGAMP hydrolase regulate the immune response?
By degrading cGAMP, it terminates STING activation and prevents excessive type I interferon production [2, 4].
What diseases are associated with cGAMP hydrolase dysfunction?
Autoinflammatory diseases, cancer, and cardiovascular disorders have been linked to dysregulated cGAMP hydrolysis [3, 4, 8].
How can I study cyclic-GMP-AMP hydrolase activity in the lab?
Use LC-MS to measure cGAMP, reporter assays for STING activation, and CRISPR knockout models [2, 7].
What is the role of ENPP1 in cGAMP hydrolysis?
ENPP1 is a major enzyme that hydrolyzes cGAMP to AMP and GMP, thereby limiting STING signaling [2, 7].
Can CRISPR be used to study cGAMP hydrolase?
Yes, CRISPR knockout, point mutation, and knock-in models are powerful tools to dissect hydrolase function [2, 4, 7].
What is the connection between cGAMP hydrolase and cancer?
Inhibiting cGAMP hydrolysis may enhance anti-tumor immunity and improve radiotherapy responses [4, 6, 7].
How does TREX1 relate to cGAMP hydrolase activity?
TREX1 degrades cytosolic DNA, reducing cGAMP production and indirectly affecting hydrolase activity.
What methods measure cGAMP levels?
Liquid chromatography-mass spectrometry (LC-MS) is the gold standard for quantifying cGAMP and its hydrolysis products [2, 7].
Conclusion
GO:0106177, cyclic-GMP-AMP hydrolase activity, is a pivotal molecular function that controls the duration and intensity of cGAS-STING signaling. Its dysregulation contributes to autoinflammation, cancer, and cardiovascular disease, making it an attractive therapeutic target [2, 4, 7]. Understanding the enzymes and regulatory mechanisms behind this activity requires precise genetic and biochemical tools, which are increasingly accessible through CRISPR technologies [2, 7].
References
- 1. Crossley MP et al.. 2023. R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response.. Nature 613(7942):187-194 PMID: 36544021
- 2. Gui X et al.. 2019. Autophagy induction via STING trafficking is a primordial function of the cGAS pathway.. Nature 567(7747):262-266 PMID: 30842662
- 3. Dou Z et al.. 2017. Cytoplasmic chromatin triggers inflammation in senescence and cancer.. Nature 550(7676):402-406 PMID: 28976970
- 4. Vanpouille-Box C et al.. 2017. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity.. Nat Commun 8:15618 PMID: 28598415
- 5. Ye R et al.. 2026. STING-NF-κB signaling builds an influenza spillover barrier.. Science 391(6788):eads4405 PMID: 41747053
- 6. McLaughlin M et al.. 2020. Inflammatory microenvironment remodelling by tumour cells after radiotherapy.. Nat Rev Cancer 20(4):203-217 PMID: 32161398
- 7. Li L et al.. 2026. Ubiquitination-directed cytosolic DNA degradation governs cGAS-STING-mediated immune response to DNA damage.. Cancer Cell 44(2):306-320.e7 PMID: 41512867
- 8. Zhang W et al.. 2025. Double-stranded DNA enhances platelet activation, thrombosis, and myocardial injury via cyclic GMP-AMP synthase.. Cardiovasc Res 121(2):353-366 PMID: 39302147
