GO:0140808 NAD+-protein-tyrosine ADP-ribosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140808 describes the enzymatic transfer of an ADP-ribose moiety from NAD+ onto a tyrosine residue of a target protein, releasing nicotinamide and H+.
• This activity is a mono-ADP-ribosylation reaction, distinct from poly-ADP-ribosylation, and is catalyzed by members of the ADP-ribosyltransferase (ART) family, including PARP7, PARP15, and bacterial toxins.
• Tyrosine-specific ADP-ribosylation regulates protein stability, immune signaling, and host-pathogen interactions, with PARP7 and AHR being marked for degradation by this modification.
• Bacterial effectors such as Clostridium difficile toxins and Pseudomonas aeruginosa ExoY exploit this activity to disrupt host cellular processes.
• Studying GO:0140808 requires tools like NAD+ analogs, activity inhibitors, and CRISPR-based gene editing to dissect enzyme-substrate relationships.
• Dysregulation of tyrosine ADP-ribosylation is implicated in cancer, infectious diseases, and inflammatory disorders, making it a target for therapeutic intervention.
Description
NAD+-protein-tyrosine ADP-ribosyltransferase activity (GO:0140808) is a molecular function that catalyzes the transfer of an ADP-ribose unit from NAD+ to the hydroxyl group of a tyrosine residue on a target protein, producing nicotinamide, a proton, and O-(ADP-D-ribosyl)-L-tyrosyl-[protein]. This post-translational modification, known as tyrosine mono-ADP-ribosylation, is emerging as a critical regulator of protein function, stability, and interactions in diverse biological contexts. Unlike poly-ADP-ribosylation, which forms long chains, this activity adds a single ADP-ribose moiety, often acting as a reversible switch in signaling pathways. Researchers are increasingly interested in GO:0140808 because it links NAD+ metabolism to immune defense, bacterial pathogenesis, and cancer biology. The enzymatic activity is mediated by ADP-ribosyltransferase (ART) domains found in both eukaryotic and bacterial proteins, and its dysregulation can lead to disease. Understanding this term at the molecular level is essential for developing targeted therapies and for interpreting genome-wide screens that identify ADP-ribosylation substrates.
NAD+-protein-tyrosine ADP-ribosyltransferase activity At A Glance
| GO ID | GO:0140808 |
|---|---|
| GO term | NAD+-protein-tyrosine ADP-ribosyltransferase activity |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Catalyzes the transfer of ADP-ribose from NAD+ to tyrosine residues on proteins, a post-translational modification |
| Reaction | L-tyrosyl-[protein] + NAD+ = H+ + nicotinamide + O-(ADP-D-ribosyl)-L-tyrosyl-[protein] |
| Enzyme family | ADP-ribosyltransferase (ART) domain-containing proteins, including PARP family members and bacterial toxins |
| Subcellular location | Cytoplasm, nucleus, and extracellular milieu depending on the enzyme |
| Related diseases | Cancer, bacterial infections, inflammatory disorders |
What Is GO:0140808?
GO:0140808 is defined by the QuickGO as the catalysis of the reaction: L-tyrosyl-[protein] + NAD+ = H+ + nicotinamide + O-(ADP-D-ribosyl)-L-tyrosyl-[protein]. In simpler terms, it is an enzymatic activity that attaches a single ADP-ribose molecule to a tyrosine amino acid on a protein, using NAD+ as the donor and releasing nicotinamide as a byproduct. This reaction is a type of mono-ADP-ribosylation and is specific for tyrosine residues, distinguishing it from serine or arginine ADP-ribosylation.
Why Is NAD+-protein-tyrosine ADP-ribosyltransferase activity Important in Cell Biology?
GO:0140808 is important because tyrosine mono-ADP-ribosylation is a reversible post-translational modification that controls key signaling proteins, often by altering their stability or interactions. This activity is exploited by bacterial pathogens to subvert host immunity, and its dysregulation in humans is linked to cancer and immune disorders. Understanding this function provides insights into NAD+ biology and offers opportunities for therapeutic targeting.
• Regulates protein stability: PARP7-mediated ADP-ribosylation marks proteins like AHR for degradation, impacting xenobiotic responses.
• Modulates immune signaling: Tyrosine ADP-ribosylation by bacterial toxins such as Pertussis toxin disrupts G-protein signaling.
• Contributes to host-pathogen interactions: Clostridium difficile toxins use this activity to impair Rho GTPases.
• Involved in antiviral defense: An mRNA ADP-ribosyltransferase blocks translation as an anti-viral strategy.
• Affects cGMP signaling: Pseudomonas aeruginosa ExoY activates cGMP signaling through ADP-ribosylation.
• Potential cancer target: PARP15 dimerization regulates its ART activity, suggesting a role in cancer cell signaling.
• Enables chemical biology: Nonhydrolyzable NAD+ analogs facilitate studying this activity.
• Guides drug discovery: Inhibitors of Pertussis toxin ART activity are being developed.
• Reveals crosstalk with ubiquitin pathway: ADP-ribosylation marks proteins for degradation via ubiquitin blockade.
• Provides tools for functional genomics: CRISPR screens can identify genes regulating this modification.
What Happens During NAD+-protein-tyrosine ADP-ribosyltransferase activity?
Substrate Recognition and Binding
In simple terms: The enzyme first grabs NAD+ and the target protein.
The ART domain of the enzyme binds NAD+ and a specific tyrosine-containing protein substrate. This step is highly regulated, as seen in PARP15 where dimerization of the ART domain controls activity. Bacterial toxins like Clostridium difficile toxin B recognize host Rho GTPases as substrates.
Catalysis and ADP-Ribose Transfer
In simple terms: The enzyme cuts NAD+ and attaches the ADP-ribose part to the tyrosine.
The enzyme cleaves the glycosidic bond of NAD+, releasing nicotinamide and transferring the ADP-ribose moiety to the tyrosine hydroxyl group, forming O-(ADP-D-ribosyl)-L-tyrosyl-[protein]. This reaction is a mono-ADP-ribosylation, distinct from polymerization.
Post-Modification Effects
In simple terms: The modified protein changes its behavior.
ADP-ribosylation on tyrosine can alter protein-protein interactions, enzymatic activity, or stability. For example, PARP7-mediated ADP-ribosylation marks AHR for degradation via the ubiquitin-proteasome system. In antiviral defense, mRNA ADP-ribosylation blocks translation.
Reversal and Regulation
In simple terms: The modification can be removed or controlled.
While specific erasers for tyrosine ADP-ribosylation are not fully characterized, the reversibility is implied by the dynamic nature of the modification. Regulation occurs at the enzyme level, such as through dimerization of PARP15 ART domains or by inhibitors like those targeting Pertussis toxin.
Key Genes Involved in GO:0140808 NAD+-protein-tyrosine ADP-ribosyltransferase activity
The following genes encode enzymes or substrates directly implicated in NAD+-protein-tyrosine ADP-ribosyltransferase activity, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PARP7 (TIPARP) | Mono-ADP-ribosyltransferase that marks proteins like AHR for degradation | Studied for its role in xenobiotic response and cancer |
| PARP15 | ART domain-containing enzyme regulated by dimerization | Model for understanding ART domain regulation |
| ARTD1 (PARP1) | Poly-ADP-ribosyltransferase with mono-ADP-ribosylation activity | Broad roles in DNA repair and transcription |
| Pertussis toxin S1 | ADP-ribosylates G-proteins | Target for inhibitor discovery |
| Clostridium difficile toxin A | Glucosyltransferase and ADP-ribosyltransferase | Key virulence factor in C. difficile infection |
| Clostridium difficile toxin B | ADP-ribosylates Rho GTPases | Studied for pathogenesis mechanisms |
| ExoY | Pseudomonas aeruginosa effector with ADP-ribosyltransferase activity | Activates cGMP signaling during infection |
| AHR | Substrate of PARP7-mediated ADP-ribosylation | Links ADP-ribosylation to xenobiotic signaling |
| RhoA | Substrate for bacterial ADP-ribosylation | Model for toxin-host interactions |
| Gαi | Substrate for Pertussis toxin | Studied in G-protein signaling |
| NAD+ | Cofactor and substrate donor | Central to the reaction |
| Ubiquitin | Pathway crosstalk with ADP-ribosylation | Marks ADP-ribosylated proteins for degradation |
| mRNA | Target of ADP-ribosylation in antiviral defense | Novel regulatory mechanism |
| ART domain | Catalytic module | Common to all enzymes in this family |
| Nicotinamide | Byproduct of the reaction | Can be used to monitor activity |
| ADP-ribose | Transferred moiety | Key modification group |
How Is NAD+-protein-tyrosine ADP-ribosyltransferase activity Regulated?
The activity of NAD+-protein-tyrosine ADP-ribosyltransferases is regulated at multiple levels. For PARP15, dimerization of the ART domain is required for catalytic activity, providing a switch for regulation. In bacterial systems, toxin activity can be controlled by environmental signals or host factors, as seen with Pseudomonas aeruginosa ExoY activation during infection. Additionally, small molecule inhibitors can modulate activity, such as compounds that inhibit Pertussis toxin. The availability of NAD+ also influences the reaction rate, linking this activity to cellular metabolism.
NAD+-protein-tyrosine ADP-ribosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PARP7 | Cancer, xenobiotic response | Knockout cell lines, overexpression models |
| PARP15 | Cancer signaling | Point mutation of dimerization interface |
| Pertussis toxin S1 | Whooping cough | Inhibitor screening assays |
| Clostridium difficile toxin B | C. difficile infection | Knockout of Rho GTPases in cells |
| ExoY | P. aeruginosa infection | Knock-in of ExoY in mammalian cells |
Cancer
ADP-ribosylation by PARP family members is implicated in cancer through regulation of protein stability and DNA repair. PARP7-mediated ADP-ribosylation of AHR leads to its degradation, affecting xenobiotic and carcinogen responses. PARP15 activity, regulated by dimerization, may influence cancer cell signaling.
Infectious Diseases
Bacterial pathogens utilize ADP-ribosyltransferases to disrupt host cellular functions. Clostridium difficile toxins ADP-ribosylate Rho GTPases, leading to cytoskeletal disruption and disease. Pertussis toxin ADP-ribosylates G-proteins, contributing to whooping cough pathogenesis. Pseudomonas aeruginosa ExoY modulates cGMP signaling during infection.
Antiviral Defense
An mRNA ADP-ribosyltransferase blocks translation as a defense mechanism against viruses, highlighting a role in innate immunity.
From NAD+-protein-tyrosine ADP-ribosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PARP7 knockout affect AHR stability? | CRISPR knockout of PARP7 in cell lines |
| How does PARP15 dimerization regulate activity? | Point mutations in ART domain |
| Can we track ADP-ribosylation in live cells? | Knock-in of tagged substrates |
| What is the role of ExoY in cGMP signaling? | Overexpression of ExoY in epithelial cells |
| Can inhibitors block Pertussis toxin activity? | In vitro enzymatic assays with inhibitors |
| Is mRNA ADP-ribosylation antiviral? | Knockout of the transferase in viral infection models |
How to Study the NAD+-protein-tyrosine ADP-ribosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro ADP-ribosylation assay | Enzyme activity using NAD+ and substrate | Screening inhibitors or testing mutants |
| Mass spectrometry | Identification of modified tyrosine residues | Mapping sites on target proteins |
| CRISPR knockout screen | Genes affecting ADP-ribosylation | Discovering regulators |
| Western blot with anti-ADP-ribose | Levels of modification | Validating substrate modification |
| Fluorescence microscopy | Subcellular localization of modification | Live-cell imaging |
| NAD+ analog labeling | Enzyme-substrate adducts | Chemical biology |
| Inhibitor screening | Compounds blocking activity | Drug discovery |
| cGMP biosensor assay | cGMP levels | Studying ExoY signaling |
Biochemical Assays
In vitro assays using recombinant ART domains and NAD+ analogs can measure ADP-ribosyltransferase activity. Nonhydrolyzable NAD+ analogs are valuable tools for trapping reaction intermediates. Inhibitor screening can identify small molecules that block activity, as shown for Pertussis toxin.
Proteomics and Mass Spectrometry
Mass spectrometry can identify ADP-ribosylated tyrosine residues on target proteins. This approach has been used to map sites on AHR and other substrates. Quantitative proteomics can assess changes in modification upon enzyme knockout or inhibition.
CRISPR Screens
Genome-wide CRISPR knockout screens can identify genes that regulate ADP-ribosylation. For example, screens have revealed crosstalk with the ubiquitin pathway. These screens are powerful for discovering novel components of the modification machinery.
Imaging and Cell-Based Assays
Fluorescently tagged ADP-ribose binding domains or antibodies can visualize ADP-ribosylation in cells. Live-cell imaging of cGMP signaling has been used to study ExoY activity. These methods provide spatial and temporal resolution.
How CRISPR Can Be Used to Study GO:0140808 NAD+-protein-tyrosine ADP-ribosyltransferase activity
Knockout
CRISPR knockout of genes encoding ADP-ribosyltransferases (e.g., PARP7, PARP15) or their substrates (e.g., AHR) can reveal loss-of-function phenotypes. For instance, PARP7 knockout stabilizes AHR, confirming its role in degradation. Knockout of bacterial toxin genes in model organisms can attenuate virulence.
Point Mutation
Introducing point mutations in the catalytic ART domain or in substrate tyrosine residues can dissect mechanism. For example, mutating the dimerization interface of PARP15 abolishes activity. Point mutations in Rho GTPases can prevent toxin-mediated ADP-ribosylation.
Knock-in
Knock-in of tagged versions of enzymes or substrates (e.g., HA-tagged PARP7) allows for affinity purification and localization studies. Knock-in of NAD+ biosensors can monitor cofactor levels.
Overexpression
Overexpression of ADP-ribosyltransferases (e.g., ExoY) in mammalian cells can mimic infection and amplify signaling effects. Overexpression of PARP15 can be used to study its regulation.
How EDITGENE Supports NAD+-protein-tyrosine ADP-ribosyltransferase activity Research
Researchers studying NAD+-protein-tyrosine ADP-ribosyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in the modification, how mutations affect enzyme function, or how the modification influences disease phenotypes. EDITGENE provides comprehensive CRISPR-based services to address these questions with precision and scale.
Contact EDITGENE today to design your custom CRISPR model for NAD+-protein-tyrosine ADP-ribosyltransferase activity research.
Frequently Asked Questions About NAD+-protein-tyrosine ADP-ribosyltransferase activity
What is NAD+-protein-tyrosine ADP-ribosyltransferase activity?
It is an enzymatic activity (GO:0140808) that transfers ADP-ribose from NAD+ to a tyrosine residue on a protein, releasing nicotinamide and H+.
What genes are involved in NAD+-protein-tyrosine ADP-ribosyltransferase activity?
Key genes include PARP7, PARP15, and bacterial toxin genes such as Pertussis toxin S1 and Clostridium difficile toxin B.
What is the difference between mono- and poly-ADP-ribosylation?
Mono-ADP-ribosylation adds a single ADP-ribose unit, while poly-ADP-ribosylation forms chains; GO:0140808 specifically describes mono-ADP-ribosylation on tyrosine.
How is NAD+-protein-tyrosine ADP-ribosyltransferase activity regulated?
Regulation occurs via enzyme dimerization (e.g., PARP15), inhibitor binding, and NAD+ availability.
What diseases are associated with this activity?
It is linked to cancer, bacterial infections (e.g., whooping cough, C. difficile), and antiviral defense.
How can I study this activity in the lab?
Use in vitro assays with NAD+ analogs, mass spectrometry, CRISPR screens, and imaging techniques.
What are the substrates of this activity?
Substrates include AHR, Rho GTPases, G-proteins, and mRNA, depending on the enzyme.
Can CRISPR be used to study this activity?
Yes, knockout, point mutation, knock-in, and overexpression models enable functional dissection.
What is the role of PARP7 in this activity?
PARP7 mono-ADP-ribosylates AHR, marking it for degradation via the ubiquitin-proteasome system.
Are there inhibitors of this activity?
Yes, compounds inhibiting Pertussis toxin ADP-ribosyltransferase activity have been discovered.
Conclusion
NAD+-protein-tyrosine ADP-ribosyltransferase activity (GO:0140808) is a fundamental enzymatic function that regulates protein fate and signaling through mono-ADP-ribosylation of tyrosine residues. Its roles in bacterial pathogenesis, cancer, and antiviral defense underscore its biomedical importance. Advances in CRISPR-based models and chemical tools are accelerating our understanding of this modification, offering new avenues for therapeutic intervention. Continued research into this activity will likely reveal additional substrates and regulatory mechanisms, further integrating NAD+ biology with disease pathways.
References
- 1. Sakari M et al.. 2025. ADP-ribosyltransferase-based biocatalysis of nonhydrolyzable NAD+ analogs.. J Biol Chem 301(1):108106 PMID: 39706271
- 2. Aktories K et al.. 2017. Clostridium difficile Toxin Biology.. Annu Rev Microbiol 71:281-307 PMID: 28657883
- 3. Gorelik A et al.. 2026. Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation.. EMBO J 45(1):261-277 PMID: 41326691
- 4. Ebenwaldner C et al.. 2025. Regulation of ADP-ribosyltransferase activity by ART domain dimerization in PARP15.. Nat Commun 16(1):9567 PMID: 41162413
- 5. Vassallo CN et al.. 2024. Anti-viral defence by an mRNA ADP-ribosyltransferase that blocks translation.. Nature 636(8041):190-197 PMID: 39443800
- 6. Hutin D et al.. 2018. Methods to Study TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP) Mono-ADP-Ribosyltransferase Activity.. Methods Mol Biol 1813:109-124 PMID: 30097864
- 7. Ashok Y et al.. 2020. Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin.. ACS Infect Dis 6(4):588-602 PMID: 31899865
- 8. Deruelle V et al.. 2025. Interplay between T3SS effectors, ExoY activation, and cGMP signaling in Pseudomonas aeruginosa infection.. Nat Commun 17(1):69 PMID: 41330935
