GO:0140806 NAD+-protein-aspartate ADP-ribosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140806 describes the enzymatic transfer of an ADP-ribose moiety from NAD+ onto the side-chain carboxyl group of an aspartate residue in a target protein, releasing nicotinamide.
• This activity is a mono-ADP-ribosylation reaction, distinct from poly-ADP-ribosylation, and is catalysed by members of the ADP-ribosyltransferase (ART) superfamily, including PARP enzymes and bacterial toxins.
• Aspartate-specific ADP-ribosylation can alter protein function, stability, and interactions, and is increasingly recognised in host defence, bacterial pathogenesis, and cancer biology.
• Key experimental approaches include NAD+ analog-based biocatalysis, mass spectrometry, and CRISPR-engineered cell models to map substrate specificity and physiological consequences.
• Dysregulation of aspartate ADP-ribosylation is linked to infectious diseases (e.g., Clostridium difficile, pertussis, Pseudomonas aeruginosa) and to cancer-associated pathways involving PARP7 and AHR.
• EDITGENE provides CRISPR knockout, point-mutation, knock-in, overexpression, and library screening services to dissect the function of GO:0140806-related genes in relevant cell models.
Description
NAD+-protein-aspartate ADP-ribosyltransferase activity (GO:0140806) is a molecular function that catalyses the transfer of an ADP-ribose unit from NAD+ to the carboxylate side chain of an aspartate residue within a target protein, producing a 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein] modification and nicotinamide. This reaction belongs to the broader family of ADP-ribosylation events, which are reversible post-translational modifications that regulate protein function, localisation, and stability. Unlike poly-ADP-ribosylation, which builds long chains, GO:0140806 specifically describes mono-ADP-ribosylation at aspartate, a modification that can be catalysed by certain PARP-family enzymes and bacterial ADP-ribosyltransferases. The importance of this activity spans both host and pathogen biology. In bacterial pathogens such as Clostridium difficile, pertussis toxin, and Pseudomonas aeruginosa, aspartate-directed ADP-ribosylation of host proteins is a key virulence mechanism that disrupts cellular signalling. In eukaryotic cells, enzymes such as PARP7 and PARP15 use this activity to regulate immune signalling, protein degradation, and antiviral defence. The modification can also be reversed by ADP-ribosylhydrolases, adding a dynamic layer of regulation. For researchers, GO:0140806 represents a focal point for understanding how NAD+ metabolism intersects with protein function in health and disease. The development of nonhydrolyzable NAD+ analogs and chemical inhibitors has enabled precise interrogation of this activity, while CRISPR-based models allow causal testing of candidate enzymes and substrates. This article synthesises current knowledge on the mechanism, key genes, disease relevance, and experimental strategies for studying NAD+-protein-aspartate ADP-ribosyltransferase activity.
NAD+-protein-aspartate ADP-ribosyltransferase activity At A Glance
| GO ID | GO:0140806 |
|---|---|
| GO term | NAD+-protein-aspartate ADP-ribosyltransferase activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Transfer of ADP-ribose from NAD+ to an aspartate residue on a target protein, releasing nicotinamide |
| Reaction | L-aspartyl-[protein] + NAD+ = 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein] + nicotinamide |
| Substrate | NAD+ and L-aspartyl-[protein] |
| Product | 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein] and nicotinamide |
| Related activity | Mono-ADP-ribosylation, distinct from poly-ADP-ribosylation |
| Representative enzymes | PARP7, PARP15, TIPARP, bacterial ART toxins |
What Is GO:0140806?
In simple terms, GO:0140806 is the enzyme activity that attaches a single ADP-ribose molecule to an aspartate amino acid on a protein. The official definition is: Catalysis of the reaction: L-aspartyl-[protein] + NAD+ = 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein] + nicotinamide. This is a mono-ADP-ribosylation event that modifies the target protein's aspartate residue, potentially altering its charge, structure, or interactions.
Why Is NAD+-protein-aspartate ADP-ribosyltransferase activity Important in Cell Biology?
NAD+-protein-aspartate ADP-ribosyltransferase activity is important because it represents a specific and reversible post-translational modification that can rapidly alter protein function in response to cellular signals or infection. This activity is exploited by bacterial pathogens to disable host defence proteins, and it is used by eukaryotic cells to regulate immune responses, protein turnover, and antiviral defence. Understanding GO:0140806 provides mechanistic insight into infectious disease, cancer, and innate immunity, and offers opportunities for therapeutic intervention through inhibitors or engineered NAD+ analogs.
• Mediates host-pathogen interactions by modifying host proteins with ADP-ribose, a key virulence strategy for Clostridium difficile, pertussis toxin, and Pseudomonas aeruginosa.
• Regulates protein stability and degradation, as shown for PARP7 and AHR, linking the activity to cancer and immune signalling.
• Contributes to antiviral defence by blocking translation through mRNA ADP-ribosylation, highlighting a role in innate immunity.
• Serves as a target for chemical inhibitors that block ADP-ribosyltransferase activity, with potential for anti-infective and anticancer therapies.
• Enables the design of nonhydrolyzable NAD+ analogs for biocatalysis and substrate profiling.
• Involved in the regulation of transcription factors such as TIPARP, which modulates aryl hydrocarbon receptor signalling.
• Provides a mechanism for crosstalk between NAD+ metabolism and cellular stress responses.
• Can be studied using CRISPR-engineered cell models to establish causal roles of specific ART enzymes.
• Dysregulation may contribute to inflammatory diseases and cancer progression through altered protein ADP-ribosylation.
• Offers a paradigm for understanding mono-ADP-ribosylation versus poly-ADP-ribosylation in cell signalling.
What Happens During NAD+-protein-aspartate ADP-ribosyltransferase activity?
Substrate recognition and NAD+ binding
In simple terms: The enzyme first grabs NAD+ and finds the target protein's aspartate residue.
The catalytic cycle begins with binding of NAD+ in the enzyme's active site, followed by recognition of a target protein containing an aspartate residue in a suitable structural context. For bacterial toxins such as pertussis toxin, specific host proteins like G proteins are recognised through a combination of sequence and structural features. In PARP-family enzymes, the ART domain mediates NAD+ binding and substrate selection, with dimerisation of the ART domain regulating activity in PARP15.
Catalysis and ADP-ribose transfer
In simple terms: The enzyme cuts NAD+ and attaches the ADP-ribose part to the aspartate on the target protein.
The catalytic step involves cleavage of the glycosidic bond between nicotinamide and ADP-ribose, releasing nicotinamide and forming a covalent bond between the ADP-ribose moiety and the target aspartate carboxyl group. This results in the formation of 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein]. The reaction is highly specific for aspartate, distinguishing it from serine or arginine ADP-ribosylation. Nonhydrolyzable NAD+ analogs can be used to trap or visualise this transfer.
Post-modification effects on target proteins
In simple terms: Once modified, the target protein may change its shape, activity, or stability.
ADP-ribosylation of aspartate can alter the target protein's charge and conformation, affecting interactions with partners or its half-life. For example, ADP-ribosylation of PARP7 and AHR marks them for degradation, linking the modification to protein turnover. In bacterial infection, modification of host proteins such as Rho GTPases disrupts signalling and cytoskeletal dynamics.
Reversal and regulation
In simple terms: Other enzymes can remove the ADP-ribose tag, making the modification reversible.
The modification is reversible through the action of ADP-ribosylhydrolases, which cleave the ADP-ribose-aspartate bond. This reversibility allows dynamic regulation of protein function. Additionally, the activity of ART enzymes can be regulated by dimerisation, as shown for PARP15, and by interacting proteins or post-translational modifications. In the context of infection, bacterial effectors like ExoY are activated by host factors, adding another layer of control.
Key Genes Involved in GO:0140806 NAD+-protein-aspartate ADP-ribosyltransferase activity
The following genes encode enzymes or substrates directly implicated in NAD+-protein-aspartate ADP-ribosyltransferase activity or its regulation, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PARP7 (TIPARP) | Mono-ADP-ribosyltransferase that modifies itself and AHR, marking them for degradation | Cancer, immune signalling, protein turnover |
| PARP15 | ART domain dimerisation regulates ADP-ribosyltransferase activity | Structural basis of ART regulation |
| ARTD1 (PARP1) | Poly-ADP-ribosyltransferase with mono-ADP-ribosylation activity on aspartate | DNA repair, cancer |
| ARTD2 (PARP2) | ADP-ribosyltransferase involved in DNA damage response | Cancer, genome stability |
| Pertussis toxin S1 | ADP-ribosylates G proteins at aspartate, disrupting signalling | Bacterial pathogenesis, vaccine development |
| C. difficile toxin A/B | Glucosyltransferase and ADP-ribosyltransferase activities | Infectious disease, toxin biology |
| ExoY | Pseudomonas aeruginosa effector with ADP-ribosyltransferase activity | Host-pathogen interaction, cGMP signalling |
| AHR | Substrate of PARP7 ADP-ribosylation, leading to degradation | Toxicology, immune regulation |
| Gαi | Substrate of pertussis toxin ADP-ribosylation | G protein signalling |
| RhoA | Substrate of bacterial ADP-ribosylation, affecting cytoskeleton | Cytoskeletal regulation |
| NAD+ | Essential cofactor for the reaction | Metabolic regulation |
| ADP-ribosylhydrolase | Reverses aspartate ADP-ribosylation | Modification dynamics |
| TIPARP | Alternative name for PARP7, regulates AHR | Gene regulation |
| ARTD17 (PARP17) | Mono-ADP-ribosyltransferase with roles in cell survival | Cancer, stress response |
| ARTD10 (PARP10) | Mono-ADP-ribosyltransferase involved in NF-κB signalling | Inflammation, cancer |
| ARTD8 (PARP8) | Mono-ADP-ribosyltransferase with unknown substrates | Enzyme specificity |
| ARTD14 (PARP14) | Mono-ADP-ribosyltransferase in immune responses | Immunology, cancer |
| ARTD9 (PARP9) | Mono-ADP-ribosyltransferase in interferon signalling | Antiviral defence |
How Is NAD+-protein-aspartate ADP-ribosyltransferase activity Regulated?
NAD+-protein-aspartate ADP-ribosyltransferase activity is regulated at multiple levels. Enzyme activity can be controlled by dimerisation of the ART domain, as demonstrated for PARP15, where dimerisation modulates catalytic efficiency. Substrate availability and NAD+ levels also influence the reaction, linking the activity to cellular metabolism. In bacterial systems, toxin activation often requires host cofactors; for example, ExoY is activated by calmodulin and actin, and its activity affects cGMP signalling. Additionally, the modification is reversible through ADP-ribosylhydrolases, which remove ADP-ribose from aspartate residues and thus terminate the signal. Post-translational modifications and interacting proteins further fine-tune the activity of enzymes like PARP7 and TIPARP.
NAD+-protein-aspartate ADP-ribosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PARP7 (TIPARP) | Cancer, protein degradation, AHR signalling | Knockout and overexpression in cancer cell lines |
| Pertussis toxin S1 | Whooping cough, G protein dysregulation | Bacterial toxin expression in mammalian cells |
| C. difficile toxins | Pseudomembranous colitis, cytoskeletal disruption | Toxin treatment of intestinal epithelial cells |
| ExoY | Pseudomonas aeruginosa infection, cGMP signalling | Infection models with ExoY mutants |
| PARP15 | Cancer, immune regulation | Knockout and point-mutation of ART domain |
Bacterial infections and toxin-mediated disease
Several bacterial pathogens use NAD+-protein-aspartate ADP-ribosyltransferase activity to modify host proteins and cause disease. Clostridium difficile toxins A and B possess ADP-ribosyltransferase activity that disrupts host cell signalling, contributing to antibiotic-associated diarrhoea and pseudomembranous colitis. Pertussis toxin from Bordetella pertussis ADP-ribosylates G proteins at aspartate, leading to whooping cough. Pseudomonas aeruginosa ExoY is a type III secretion effector with ADP-ribosyltransferase activity that alters cGMP signalling during infection. Inhibitors of these enzymes are being explored as anti-infective agents.
Cancer and protein degradation
ADP-ribosylation of aspartate residues can mark proteins for degradation, as shown for PARP7 and AHR. This links GO:0140806 to cancer biology, where dysregulated protein turnover contributes to tumour progression. PARP7 is a mono-ADP-ribosyltransferase that modifies itself and AHR, leading to their degradation via the ubiquitin-proteasome system. Targeting PARP7 activity is an emerging strategy in oncology.
Antiviral defence and innate immunity
An mRNA ADP-ribosyltransferase that blocks translation has been shown to provide antiviral defence, highlighting a role for ADP-ribosylation in innate immunity. This activity modifies mRNA, but the broader concept of ADP-ribosylation as a defence mechanism is relevant to GO:0140806. PARP9 and PARP14, which have mono-ADP-ribosyltransferase activity, are involved in interferon signalling and antiviral responses.
From NAD+-protein-aspartate ADP-ribosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of PARP7 affect AHR stability? | PARP7 knockout cell line |
| How does ART domain dimerisation regulate PARP15 activity? | Point mutations disrupting dimer interface |
| Can nonhydrolyzable NAD+ analogs trap enzyme-substrate complexes? | Knock-in of tagged substrate with analog treatment |
| What is the role of ExoY ADP-ribosylation in infection? | Overexpression of ExoY in epithelial cells |
| Does ADP-ribosylation of G proteins alter signalling? | Knock-in of aspartate-to-glutamate mutant G protein |
| Can CRISPR screening identify new substrates? | Genome-wide knockout library with ADP-ribosylation readout |
How to Study the NAD+-protein-aspartate ADP-ribosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro ADP-ribosylation assay | Enzyme activity using NAD+ analogs | Kinetic analysis of ART enzymes |
| Mass spectrometry | Identification of modified aspartate residues | Substrate mapping |
| CRISPR knockout screen | Genes required for ADP-ribosylation | Discovery of regulatory pathways |
| Western blot with anti-ADP-ribose | Levels of ADP-ribosylated proteins | Drug or genetic perturbation |
| Fluorescence microscopy | Localisation of ADP-ribosylation | Cellular imaging |
| NAD+ analog pull-down | Enzyme-substrate complexes | Biocatalysis and profiling |
| Bacterial toxin treatment | Host protein modification | Infection biology |
| CRISPR point mutation | Effect of specific aspartate mutation | Causal testing of modification sites |
Biochemical assays for ADP-ribosyltransferase activity
In vitro assays using recombinant enzymes and NAD+ or nonhydrolyzable NAD+ analogs can directly measure GO:0140806 activity. These assays often employ radioactive NAD+ or fluorescent analogs to detect ADP-ribose incorporation into substrate proteins. For bacterial toxins, specific substrates like G proteins or Rho GTPases are used.
Mass spectrometry and proteomics
Mass spectrometry can identify the specific aspartate residues modified by ADP-ribosylation and map substrate sites. This approach is valuable for discovering new substrates and understanding the modification's stoichiometry. Coupling with affinity purification using ADP-ribose-binding domains enriches modified proteins.
CRISPR-based genetic screens
Genome-wide CRISPR knockout or activation screens can identify genes that regulate or are required for ADP-ribosylation at aspartate. For example, screens have revealed factors in the ubiquitin pathway that influence PARP7 stability. These screens can be combined with ADP-ribosylation-specific antibodies or reporters.
Imaging and cellular localisation
Fluorescently tagged ADP-ribose-binding domains or antibodies can visualise ADP-ribosylation in cells and tissues. Live-cell imaging of tagged enzymes or substrates can reveal dynamics of the modification. This is particularly useful for studying bacterial toxin effects on host cells.
How CRISPR Can Be Used to Study GO:0140806 NAD+-protein-aspartate ADP-ribosyltransferase activity
Knockout
CRISPR knockout of genes encoding ADP-ribosyltransferases (e.g., PARP7, PARP15) or their substrates can abolish specific ADP-ribosylation events, allowing researchers to test causality. Knockout cell lines are essential for distinguishing the roles of individual enzymes in complex cellular processes.
Point Mutation
Introducing point mutations in the catalytic domain of ART enzymes (e.g., catalytic glutamate to glutamine) or in the target aspartate residue of substrate proteins can specifically disable the modification without affecting protein expression. This is critical for linking the modification to functional outcomes.
Knock-in
Knock-in of tagged versions of ART enzymes or substrates (e.g., HA-tag, GFP) enables affinity purification, imaging, and proteomic analysis of ADP-ribosylation complexes. Knock-in of nonhydrolyzable NAD+ analog-sensitive mutants can trap enzyme-substrate intermediates.
Overexpression
Overexpression of wild-type or mutant ART enzymes in cell lines can amplify ADP-ribosylation signals for detection and functional studies. This approach is useful for studying bacterial toxins like ExoY or pertussis toxin in host cells.
How EDITGENE Supports NAD+-protein-aspartate ADP-ribosyltransferase activity Research
Researchers studying NAD+-protein-aspartate ADP-ribosyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in a specific modification, disease phenotype, or signalling pathway. EDITGENE provides a comprehensive suite of CRISPR-based services to enable such investigations with high precision and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for NAD+-protein-aspartate ADP-ribosyltransferase activity research.
Frequently Asked Questions About NAD+-protein-aspartate ADP-ribosyltransferase activity
What is NAD+-protein-aspartate ADP-ribosyltransferase activity?
It is an enzymatic activity (GO:0140806) that transfers an ADP-ribose group from NAD+ to an aspartate residue on a target protein, releasing nicotinamide.
What genes are involved in NAD+-protein-aspartate ADP-ribosyltransferase activity?
Key genes include PARP7 (TIPARP), PARP15, and bacterial toxin genes such as pertussis toxin S1 and ExoY.
What is the reaction catalysed by GO:0140806?
L-aspartyl-[protein] + NAD+ = 4-O-(ADP-D-ribosyl)-L-aspartyl-[protein] + nicotinamide.
How is NAD+-protein-aspartate ADP-ribosylation different from poly-ADP-ribosylation?
It adds a single ADP-ribose unit to aspartate, whereas poly-ADP-ribosylation builds long chains on other residues.
Which diseases are linked to this activity?
Bacterial infections (C. difficile, pertussis, Pseudomonas), cancer, and immune disorders.
How can I study NAD+-protein-aspartate ADP-ribosyltransferase activity?
Use in vitro assays with NAD+ analogs, mass spectrometry, CRISPR screens, and imaging.
What are the substrates of this activity?
Substrates include G proteins, AHR, PARP7 itself, and other proteins with accessible aspartate residues.
Can this activity be inhibited?
Yes, compounds inhibiting ADP-ribosyltransferase activity have been discovered, e.g., for pertussis toxin.
What is the role of PARP7 in this activity?
PARP7 is a mono-ADP-ribosyltransferase that modifies itself and AHR, marking them for degradation.
How does EDITGENE support research on GO:0140806?
EDITGENE offers CRISPR knockout, point mutation, knock-in, overexpression, and library screening services for genes related to this activity.
Conclusion
NAD+-protein-aspartate ADP-ribosyltransferase activity (GO:0140806) is a specialised post-translational modification with critical roles in bacterial pathogenesis, cancer, and immunity. The reaction is catalysed by diverse enzymes, including PARP-family proteins and bacterial toxins, and is regulated by dimerisation, substrate availability, and reversibility. Understanding this activity offers insights into fundamental cell biology and potential therapeutic targets. EDITGENE provides the CRISPR tools needed to dissect the genes and mechanisms underlying this modification, empowering researchers to translate findings into new treatments.
References
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- 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
