GO:0050700 CARD domain binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0050700 CARD domain binding describes the molecular function of selectively binding to a caspase recruitment domain (CARD), a death domain-fold module that mediates homophilic protein-protein interactions.
• CARD-CARD interactions nucleate signaling platforms such as inflammasomes, which activate caspase-1 and drive IL-1beta and IL-18 maturation.
• The function is central to innate immunity, as shown by AIM2 recognizing cytosolic dsDNA and forming a caspase-1-activating inflammasome with ASC.
• CARD domain binding is not limited to animals; CARD domains also mediate anti-phage defence in bacterial gasdermin systems.
• Dysregulated CARD-dependent assembly is linked to autoinflammatory, autoimmune, and metabolic diseases, including gout and tendinopathy.
• CRISPR knockout, point-mutation, knock-in, and overexpression models are essential to test whether CARD-binding interfaces are causally required for signaling.
Description
GO:0050700 CARD domain binding is a molecular function term that captures the ability of a protein to bind a CARD (caspase recruitment domain), a compact alpha-helical protein-protein interaction module belonging to the death domain-fold superfamily. CARDs are found in the N-terminal prodomains of several caspases and in apoptosis- and inflammation-regulatory proteins, where they mediate highly specific homophilic interactions that assemble signaling complexes. Because these interactions are both selective and reversible, they provide a general mechanism for building higher-order signaling platforms in cells. The term is therefore of broad interest to researchers studying innate immunity, cell death, and inflammation. The best-characterized example is the AIM2 inflammasome, in which AIM2 recognizes cytosolic double-stranded DNA and uses its CARD to bind the CARD of ASC (PYCARD), forming a caspase-1-activating platform. This CARD-CARD nucleation event is a textbook illustration of GO:0050700 and explains why the term is annotated to multiple inflammasome adaptors and sensors. Beyond canonical inflammasomes, CARD-dependent interactions participate in NF-kB activation, mitophagy regulation, and even bacterial immunity, indicating deep evolutionary conservation of the function. Consequently, CARD domain binding is not merely a structural curiosity but a central node in host defence and inflammatory disease.
CARD domain binding At A Glance
| GO ID | GO:0050700 |
|---|---|
| GO term | CARD domain binding |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Selective binding to a CARD module to nucleate homophilic signaling complexes |
| Domain fold | Death domain-fold superfamily; six or seven anti-parallel alpha-helices |
| Representative proteins | AIM2, ASC/PYCARD, NLRC5, NLRP6, caspase-1 |
| Biological context | Innate immunity, inflammasome assembly, apoptosis, anti-phage defence |
| Disease relevance | Autoinflammation, gout, tendinopathy, neuroinflammation |
What Is GO:0050700?
In plain terms, GO:0050700 CARD domain binding means a protein physically and selectively attaches to a CARD module on another protein. The QuickGO definition specifies that the CARD is an N-terminal caspase recruitment domain, a protein-protein interaction domain of the death domain-fold superfamily, whose members share a bundle of six or seven anti-parallel alpha-helices that form highly specific homophilic interactions between signaling partners. CARDs occur in the N-terminal prodomains of several caspases and in apoptosis-regulatory proteins, and they mediate assembly of CARD-containing proteins that activate or suppress CARD-carrying caspases. Thus, the function is defined by the binding event itself, not by a catalytic activity, and it typically serves to recruit or oligomerize signaling partners.
Why Is CARD domain binding Important in Cell Biology?
CARD domain binding is important because it is the molecular switch that converts soluble sensors and adaptors into active signaling platforms. In the AIM2 inflammasome, CARD-CARD interaction between AIM2 and ASC is required for caspase-1 activation and for the release of mature IL-1beta and IL-18, which are central mediators of fever, inflammation, and host defence. The same principle applies to other CARD-containing sensors and adaptors, making GO:0050700 a recurring mechanism in innate immune signaling. Because CARD interactions are homophilic and highly specific, they offer attractive targets for therapeutic intervention in inflammatory disease. Moreover, the discovery that CARD domains mediate anti-phage defence in bacterial gasdermin systems shows that this binding function is ancient and broadly relevant across kingdoms. For researchers, understanding CARD domain binding is therefore essential for interpreting inflammasome biology, designing CRISPR models, and developing drugs that modulate inflammatory signaling.
• Defines the core assembly step of inflammasomes such as AIM2-ASC-caspase-1.
• Controls maturation of IL-1beta and IL-18, key pyrogenic and inflammatory cytokines.
• Links cytosolic DNA sensing to caspase-1 activation in infection and autoimmunity.
• Participates in mitophagy and immunity regulation, connecting inflammation to autophagy.
• Is conserved in bacteria, where CARD domains mediate anti-phage defence.
• Contributes to gout and other autoinflammatory diseases driven by innate immune activation.
• Is implicated in tendinopathy through AIM2-PYCARD/ASC stability and selective autophagy.
• Is involved in post-stroke neuroinflammation via microglial NLRC5 and lysosomal dysfunction.
• Provides a druggable interface for anti-inflammatory therapeutic development.
• Requires precise CRISPR models to distinguish causal CARD interfaces from bystander effects.
What Happens During CARD domain binding?
Sensor activation and CARD exposure
In simple terms: A sensor protein changes shape after detecting a danger signal, exposing its CARD so it can bind a partner.
In the AIM2 inflammasome, AIM2 recognizes cytosolic double-stranded DNA and then forms a caspase-1-activating inflammasome with ASC. This activation step is a prerequisite for CARD domain binding because the CARD must be available to engage a partner CARD. In other systems, phase separation drives RNA virus-induced activation of the NLRP6 inflammasome, showing that CARD-dependent assembly can be coupled to condensation of signaling components. Thus, the first stage of GO:0050700 is signal-induced availability of a CARD module.
Homophilic CARD-CARD nucleation
In simple terms: Two CARD-containing proteins lock together through their CARDs, like matching puzzle pieces, to start a signaling cluster.
The QuickGO definition emphasizes that CARDs form highly specific homophilic interactions between signaling partners. In the AIM2 pathway, the CARD of AIM2 binds the CARD of ASC, nucleating a platform that recruits caspase-1. This homophilic binding is the defining event of GO:0050700 and explains why the term is annotated to both sensors and adaptors. The interaction is not random; it depends on the death domain-fold architecture of six or seven anti-parallel alpha-helices that presents complementary surfaces.
Filament assembly and caspase-1 recruitment
In simple terms: Once the first CARDs bind, more proteins join to form a long filament that grabs and activates caspase-1.
CARD-CARD nucleation leads to assembly of higher-order filaments that recruit CARD-carrying caspases. In the AIM2 inflammasome, this assembly produces a caspase-1-activating platform, which then processes pro-IL-1beta and pro-IL-18. The QuickGO definition explicitly notes that CARD-containing proteins participate in activation or suppression of CARD-carrying caspases, so the outcome of binding can be either activating or inhibitory depending on the partners. This stage is therefore the functional readout of GO:0050700.
Regulation by autophagy and stability
In simple terms: Cells can remove or stabilize CARD-containing proteins through autophagy, tuning how much signaling occurs.
Pristimerin suppresses the AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy, demonstrating that CARD-containing complexes are subject to autophagic control. Similarly, microglial NLRC5 drives lysosomal dysfunction to disrupt autophagic flux and promote post-stroke neuroinflammation, linking CARD-domain-dependent signaling to lysosomal and autophagic regulation. These findings show that CARD domain binding is not a static event but is tuned by degradation and trafficking pathways.
Evolutionary conservation in bacteria
In simple terms: Even bacteria use CARD-like domains to fight viruses, showing this binding function is very old.
CARD domains mediate anti-phage defence in bacterial gasdermin systems, indicating that the CARD fold and its binding function predate metazoan immunity. This conservation supports the view that GO:0050700 represents a fundamental protein-interaction mechanism rather than a vertebrate-specific innovation. It also suggests that CARD-based signaling principles can be studied in simple model systems.
Key Genes Involved in GO:0050700 CARD domain binding
The following genes and proteins are central to CARD domain binding and its downstream signaling.
| Gene | Major Role | Research Relevance |
|---|---|---|
| AIM2 | Cytosolic dsDNA sensor that binds ASC via CARD to form a caspase-1-activating inflammasome | Model for DNA-sensing and autoinflammation |
| PYCARD (ASC) | Adaptor with a CARD that binds AIM2 and other sensors to nucleate inflammasomes | Core CARD-CARD interaction node |
| CASP1 | CARD-carrying caspase recruited to inflammasome platforms for activation | Effector of CARD-dependent signaling |
| NLRC5 | CARD-containing regulator linked to microglial lysosomal dysfunction and neuroinflammation | Target for post-stroke neuroinflammation studies |
| NLRP6 | Inflammasome sensor whose activation involves phase separation during RNA virus infection | Model for condensation-driven CARD signaling |
| NLRP3 | Inflammasome sensor that assembles with ASC and caspase-1 in innate immunity | Broadly studied CARD-dependent platform |
| NLRC4 | CARD-containing sensor that assembles inflammasomes in response to bacterial ligands | Model for bacterial sensing |
| BIRC2/cIAP1 | Apoptosis-regulatory protein with CARD-related signaling roles | Link between CARD binding and cell death |
| BIRC3/cIAP2 | Apoptosis-regulatory protein implicated in CARD-dependent NF-kB signaling | Target for inflammation and cancer studies |
| RIPK2 | CARD-containing kinase that mediates innate immune signaling downstream of NOD receptors | Model for CARD-dependent NF-kB activation |
| NOD1 | CARD-containing pattern recognition receptor for bacterial peptides | Study of CARD-mediated bacterial sensing |
| NOD2 | CARD-containing receptor linked to innate immune responses and inflammatory disease | Disease-relevant CARD model |
| CASP9 | CARD-carrying initiator caspase in apoptosis | Model for CARD-dependent apoptosis |
| CASP2 | CARD-containing caspase involved in stress-induced apoptosis | Study of CARD-caspase recruitment |
| GSDMB | Gasdermin family member linked to CARD-like defence systems in bacteria | Evolutionary model for CARD defence |
| GSDMD | Gasdermin executed in inflammasome-driven pyroptosis downstream of CARD signaling | Readout of CARD-dependent cell death |
| IL1B | Cytokine matured downstream of CARD-dependent caspase-1 activation | Functional readout of inflammasome activity |
| IL18 | Cytokine matured downstream of CARD-dependent caspase-1 activation | Functional readout of inflammasome activity |
How Is CARD domain binding Regulated?
CARD domain binding is regulated at multiple levels. Autophagic degradation of AIM2-PYCARD/ASC complexes can suppress inflammasome signaling, as shown by pristimerin treatment in tendinopathy models. Lysosomal dysfunction and disrupted autophagic flux in microglia can amplify NLRC5-dependent neuroinflammation, indicating that organelle quality control modulates CARD-dependent platforms. Phase separation is another regulatory layer, as RNA virus-induced activation of the NLRP6 inflammasome is driven by condensation of signaling components. In addition, mitophagy and general autophagy pathways influence immunity and autoimmune disease, providing a broader context for how CARD-containing complexes are controlled. Together, these mechanisms determine the intensity and duration of CARD domain binding-dependent signaling.
CARD domain binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| AIM2 | DNA-sensing autoinflammation and tendinopathy | Knockout and point-mutation models in macrophages |
| PYCARD (ASC) | Inflammasome-driven autoinflammatory disease | Knock-in of CARD interface mutations |
| NLRC5 | Post-stroke neuroinflammation | Microglial knockout and overexpression models |
| NLRP6 | RNA virus-induced inflammasome activation | Phase-separation reporter knock-in models |
| NLRP3 | Crystal-induced inflammation and gout | Knockout and inhibitor-treated models |
Autoinflammatory and autoimmune diseases
CARD domain binding is directly implicated in autoinflammatory and autoimmune conditions because it drives inflammasome assembly and cytokine maturation. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC, a process that can become pathogenic when dysregulated. Emerging views of mitophagy in immunity and autoimmune diseases further link CARD-dependent signaling to autoimmunity through mitochondrial quality control. Genetic and epigenetic regulation of the innate immune response to gout highlights how CARD-containing sensors contribute to urate crystal-driven inflammation.
Gout and crystal-induced inflammation
Gout is a prototypical autoinflammatory disease in which innate immune activation by monosodium urate crystals triggers IL-1beta release. Because CARD domain binding is required for inflammasome assembly and caspase-1 activation, it is mechanistically central to gout pathogenesis. Genetic and epigenetic factors that tune CARD-dependent signaling may therefore modify gout susceptibility and severity.
Tendinopathy and musculoskeletal inflammation
Pristimerin suppresses the AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy, directly connecting CARD domain binding to musculoskeletal disease. This study shows that disrupting the stability of CARD-containing complexes can reduce inflammation and tissue damage. It also supports the concept that CARD-CARD interfaces are viable therapeutic targets in tendinopathy.
Neuroinflammation and stroke
Microglial NLRC5 drives lysosomal dysfunction to disrupt autophagic flux and promote post-stroke neuroinflammation, linking CARD-containing proteins to brain injury. Because NLRC5 is a CARD-containing regulator, its effects are likely mediated in part through CARD domain binding events. This positions GO:0050700 as a potential node for therapeutic intervention in neuroinflammatory disease.
From CARD domain binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is the CARD-CARD interface required for inflammasome assembly? | Point-mutation knock-in of CARD interface residues |
| Does loss of a CARD-containing sensor reduce cytokine maturation? | CRISPR knockout of AIM2 or PYCARD |
| Can CARD-dependent signaling be tracked in live cells? | Tagged knock-in of CARD proteins with fluorescent reporters |
| Does overexpression of a CARD protein drive spontaneous inflammation? | Overexpression cell models |
| Is CARD-dependent neuroinflammation reversible? | Microglial knockout and rescue models |
| Are CARD defence systems conserved in bacteria? | Bacterial gasdermin knockout and reconstitution models |
How to Study the CARD domain binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Co-immunoprecipitation | Physical interaction between CARD-containing proteins | Validation of CARD-CARD binding |
| Mass spectrometry | Composition of CARD-dependent complexes | Discovery of new interaction partners |
| Fluorescence microscopy | Assembly and localization of CARD signaling clusters | Live-cell imaging of inflammasomes |
| IL-1beta ELISA | Functional output of caspase-1 activation | Inflammasome activity readout |
| Caspase-1 activity assay | Proteolytic activity downstream of CARD binding | Quantification of inflammasome activation |
| Autophagy flux assay | Degradation of CARD-containing complexes | Study of selective autophagy regulation |
| Lysosomal function assay | Organelle dysfunction linked to CARD signaling | Neuroinflammation models |
| Phase-separation assay | Condensation of CARD signaling components | RNA virus-induced inflammasome studies |
CRISPR knockout and point-mutation screens
CRISPR knockout of CARD-containing genes such as AIM2 or PYCARD can test whether CARD domain binding is required for inflammasome activation and cytokine release. Point-mutation knock-in of CARD interface residues allows precise dissection of homophilic binding surfaces without deleting the entire protein. These approaches are essential because CARD domains often have additional scaffolding functions that can confound complete knockouts.
Biochemical and proteomic analysis of CARD complexes
Co-immunoprecipitation and mass spectrometry can identify CARD-containing interaction partners and quantify binding stoichiometry. Because CARD-CARD interactions are homophilic and specific, crosslinking and native gel analysis can reveal filament formation. Proteomic profiling of inflammasome complexes after stimulation provides a systems-level view of CARD domain binding.
Imaging and phase-separation assays
Fluorescence microscopy of tagged CARD proteins can visualize assembly of signaling clusters in cells. Phase-separation assays, including droplet formation and FRAP, are useful because RNA virus-induced NLRP6 inflammasome activation is driven by condensation. Live-cell imaging of AIM2 and ASC recruitment provides direct evidence of CARD-dependent nucleation.
Functional readouts of inflammasome activity
IL-1beta and IL-18 ELISA, caspase-1 activity assays, and pyroptosis measurements are standard readouts of CARD-dependent signaling. Autophagy flux assays can reveal whether CARD-containing complexes are being degraded, as shown for AIM2-PYCARD/ASC stability. Lysosomal function assays are relevant for NLRC5-dependent neuroinflammation models.
How CRISPR Can Be Used to Study GO:0050700 CARD domain binding
Knockout
CRISPR knockout of CARD-containing genes such as AIM2 or PYCARD is used to test whether CARD domain binding is required for inflammasome assembly and cytokine maturation. Knockout models are also valuable for studying NLRC5-dependent neuroinflammation and for confirming that observed phenotypes are CARD-dependent. Because CARD proteins can have redundant functions, careful validation with multiple guides is recommended.
Point Mutation
Point-mutation knock-in of CARD interface residues allows researchers to disrupt homophilic binding while preserving protein expression and scaffolding functions. This is particularly important for distinguishing CARD domain binding from other domains in the same protein. Point mutants can be used in rescue experiments to test causality in inflammasome activation.
Knock-in
Tagged knock-in of CARD proteins with fluorescent or affinity tags enables live-cell imaging and biochemical purification of CARD-dependent complexes. Knock-in of disease-associated variants can model how human mutations alter CARD domain binding and signaling. These models are essential for translating genetic findings into mechanistic insight.
Overexpression
Overexpression of CARD-containing proteins can drive spontaneous inflammasome assembly and cytokine release, providing a gain-of-function system to study CARD domain binding. Overexpression models are useful for testing inhibitors that target CARD-CARD interfaces. They can also reveal phase-separation behavior when combined with imaging.
How EDITGENE Supports CARD domain binding Research
Researchers studying CARD domain binding-related genes often need to determine whether a candidate gene is causally involved in inflammasome assembly, cytokine maturation, or inflammatory disease. EDITGENE provides end-to-end CRISPR services to generate precisely engineered cell models that answer these questions.
Contact EDITGENE today to design your custom CRISPR model for CARD domain binding research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| CARD11 Knockout HEK293 Cell Line | EDJ-KQ138 | Human | 84433 | Details Get a Quote |
| CARD14 Knockout HEK293 Cell Line | EDJ-KQ546 | Human | 79092 | Details Get a Quote |
| NOD1 Knockout HEK293 Cell Line | EDJ-KQ1045 | Human | 10392 | Details Get a Quote |
| CASP1 Knockout HEK293 Cell Line | EDJ-KQ1456 | Human | 834 | Details Get a Quote |
| NOD2 Knockout HEK293 Cell Line | EDJ-KQ1486 | Human | 64127 | Details Get a Quote |
| CASP4 Knockout HEK293 Cell Line | EDJ-KQ2380 | Human | 837 | Details Get a Quote |
| IRGM Knockout HEK293 Cell Line | EDJ-KQ3168 | Human | 345611 | Details Get a Quote |
| MAVS Knockout HEK293 Cell Line | EDJ-KQ3570 | Human | 57506 | Details Get a Quote |
| RIPK2 Knockout HEK293 Cell Line | EDJ-KQ6357 | Human | 8767 | Details Get a Quote |
| CARD16 Knockout HEK293 Cell Line | EDJ-KQ7453 | Human | 114769 | Details Get a Quote |
| CARD8 Knockout HEK293 Cell Line | EDJ-KQ7727 | Human | 22900 | Details Get a Quote |
| CARD19 Knockout HEK293 Cell Line | EDJ-KQ10044 | Human | 84270 | Details Get a Quote |
| CARD9 Knockout HEK293 Cell Line | EDJ-KQ12725 | Human | 64170 | Details Get a Quote |
| CARD18 Knockout HEK293 Cell Line | EDJ-KQ12726 | Human | 59082 | Details Get a Quote |
| CARD11 Knockout HeLa Cell Line | EDJ-KQ18923 | Human | 84433 | Details Get a Quote |
Displaying Records 1 To 15 Of 63 Records
- 1
- 2
- Next Page »
Frequently Asked Questions About CARD domain binding
What is GO:0050700 CARD domain binding?
GO:0050700 is a molecular function term describing selective binding to a CARD (caspase recruitment domain), a death domain-fold module that mediates homophilic protein-protein interactions in signaling complexes.
What genes are involved in CARD domain binding?
Key genes include AIM2, PYCARD (ASC), CASP1, NLRC5, NLRP6, NLRP3, NLRC4, RIPK2, NOD1, and NOD2, all of which contain or bind CARD modules.
How does CARD domain binding activate the inflammasome?
CARD-CARD interaction between a sensor such as AIM2 and the adaptor ASC nucleates a platform that recruits and activates caspase-1, leading to IL-1beta and IL-18 maturation.
Is CARD domain binding involved in disease?
Yes, dysregulated CARD-dependent signaling is linked to autoinflammatory and autoimmune diseases, gout, tendinopathy, and post-stroke neuroinflammation.
What is the difference between a CARD and a death domain?
Both belong to the death domain-fold superfamily, but CARDs are defined by their N-terminal caspase recruitment function and their specific homophilic interaction properties.
Can CRISPR be used to study CARD domain binding?
Yes, CRISPR knockout, point-mutation knock-in, tagged knock-in, and overexpression models are widely used to dissect CARD-dependent signaling.
What experimental models are best for CARD domain binding research?
Macrophage and microglial cell lines with knockout or knock-in of CARD-containing genes are commonly used, along with bacterial models for evolutionary studies.
How is CARD domain binding regulated?
It is regulated by autophagy-mediated degradation of CARD complexes, phase separation, and lysosomal quality control pathways.
Are CARD domains conserved in bacteria?
Yes, CARD domains mediate anti-phage defence in bacterial gasdermin systems, showing deep evolutionary conservation.
What methods measure CARD domain binding?
Co-immunoprecipitation, mass spectrometry, fluorescence imaging, IL-1beta ELISA, caspase-1 activity assays, and phase-separation assays are commonly used.
Conclusion
GO:0050700 CARD domain binding defines a fundamental protein interaction that assembles signaling platforms central to innate immunity and inflammation. From the AIM2-ASC-caspase-1 inflammasome to bacterial anti-phage defence, CARD-CARD interactions provide specificity and switch-like control of inflammatory outputs. Dysregulation of these interactions contributes to autoinflammatory and autoimmune diseases, making them attractive therapeutic targets. CRISPR-based knockout, point-mutation, knock-in, and overexpression models are indispensable for testing causality and for developing interventions that modulate CARD domain binding.
References
- 1. Xu Y et al.. 2020. Emerging views of mitophagy in immunity and autoimmune diseases.. Autophagy 16(1):3-17 PMID: 30951392
- 2. Shen C et al.. 2021. Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.. Cell 184(23):5759-5774.e20 PMID: 34678144
- 3. Futosi K et al.. 2013. Neutrophil cell surface receptors and their intracellular signal transduction pathways.. Int Immunopharmacol 17(3):638-50 PMID: 23994464
- 4. Wein T et al.. 2025. CARD domains mediate anti-phage defence in bacterial gasdermin systems.. Nature 639(8055):727-734 PMID: 39880956
- 5. de Lima JD et al.. 2023. Genetic and Epigenetic Regulation of the Innate Immune Response to Gout.. Immunol Invest 52(3):364-397 PMID: 36745138
- 6. Jiang H et al.. 2024. Pristimerin suppresses AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy.. Autophagy 20(1):76-93 PMID: 37647255
- 7. Xu S et al.. 2025. Microglial NLRC5 drives lysosomal dysfunction to disrupt autophagic flux and promote post-stroke neuroinflammation.. J Neuroinflammation 22(1):253 PMID: 41174779
- 8. Hornung V et al.. 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.. Nature 458(7237):514-8 PMID: 19158675
