GO:0140807 NAD+-protein-glutamate ADP-ribosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140807 describes the enzymatic transfer of an ADP-ribose moiety from NAD+ to a glutamate residue on a target protein, releasing nicotinamide.
• This activity is carried out by ADP-ribosyltransferase (ART) domains found in bacterial toxins and human PARP family enzymes.
• Glutamate-specific ADP-ribosylation can alter protein function, stability, and interactions, and is reversible.
• Dysregulation of this activity is linked to cancer, infectious diseases, and antiviral responses.
• Key experimental approaches include acid-urea gel electrophoresis, radiolabeling, and mass spectrometry.
• CRISPR-based knockout, knock-in, and overexpression models are essential to dissect the physiological roles of these enzymes.
Description
NAD+-protein-glutamate ADP-ribosyltransferase activity (GO:0140807) is a molecular function that catalyzes the transfer of an ADP-ribose group from nicotinamide adenine dinucleotide (NAD+) to a glutamate residue on a target protein, producing a 5-O-(ADP-D-ribosyl)-L-glutamyl-[protein] modification and releasing nicotinamide. This post-translational modification, known as glutamate ADP-ribosylation, is employed by both bacterial toxins and eukaryotic enzymes to modulate protein function. The reaction is fundamental to cellular processes such as DNA damage repair, immune signaling, and host-pathogen interactions. Researchers study this activity to understand its roles in health and disease, and to develop therapeutics targeting ADP-ribosyltransferases. The specificity for glutamate distinguishes it from other ADP-ribosylation reactions that modify arginine, serine, or cysteine residues.
NAD+-protein-glutamate ADP-ribosyltransferase activity At A Glance
| GO ID | GO:0140807 |
|---|---|
| GO term | NAD+-protein-glutamate ADP-ribosyltransferase activity |
| Ontology | molecular_function |
| Synonym | None |
| Definition | Catalysis of the reaction: L-glutamyl-[protein] + NAD+ = 5-O-(ADP-D-ribosyl)-L-glutamyl-[protein] + nicotinamide. |
| Major function | Transfer of ADP-ribose from NAD+ to glutamate residues on target proteins, modulating their function. |
| EC number | Not assigned |
| Related activity | NAD+-protein-arginine ADP-ribosyltransferase activity (GO:0003956) |
What Is GO:0140807?
This term describes the catalytic activity of an enzyme that transfers the ADP-ribose moiety of NAD+ to the side-chain carboxyl group of a glutamate residue within a protein substrate. The reaction yields a glutamate-linked ADP-ribosylated protein and nicotinamide as a byproduct. This activity is a type of mono-ADP-ribosylation, as opposed to poly-ADP-ribosylation, and is mediated by enzymes containing an ADP-ribosyltransferase (ART) domain.
Why Is NAD+-protein-glutamate ADP-ribosyltransferase activity Important in Cell Biology?
Glutamate-specific ADP-ribosylation is a critical post-translational modification that regulates diverse cellular processes, including DNA repair, transcription, and immune responses. Bacterial pathogens such as Clostridium difficile and Bordetella pertussis use this activity to disrupt host cell signaling, making it a target for anti-virulence drugs. In humans, dysregulation of ADP-ribosyltransferases like PARP7 and PARP15 is implicated in cancer and autoimmune diseases. Understanding this activity at the molecular level is essential for developing selective inhibitors and for interpreting disease-associated mutations.
• Regulates protein function and stability through reversible modification.
• Plays a key role in DNA damage response and genome maintenance.
• Mediates host-pathogen interactions by bacterial toxins.
• Involved in antiviral defense mechanisms.
• Contributes to cancer progression and immune evasion.
• Target for drug discovery against bacterial infections.
• Essential for understanding PARP family enzyme biology.
• Provides a mechanism for crosstalk with ubiquitination and other modifications.
• Enables precise modulation of signaling pathways in cells.
• Facilitates development of CRISPR models to study gene function.
What Happens During NAD+-protein-glutamate ADP-ribosyltransferase activity?
Substrate Binding and NAD+ Recognition
In simple terms: The enzyme grabs NAD+ and the target protein.
The ADP-ribosyltransferase enzyme binds NAD+ in its active site and recognizes a specific glutamate residue on the target protein. Structural studies of PARP15 have shown that dimerization of the ART domain regulates this binding and subsequent catalytic activity. Bacterial toxins like C. difficile toxin A also bind NAD+ and target host proteins, though their substrate specificity differs.
Catalysis and ADP-Ribose Transfer
In simple terms: The enzyme snips NAD+ and attaches the ADP-ribose part to the protein.
The catalytic reaction involves cleavage of the glycosidic bond between nicotinamide and ADP-ribose, followed by formation of an ester bond between the ADP-ribose and the glutamate side chain. This results in the release of nicotinamide and the formation of 5-O-(ADP-D-ribosyl)-L-glutamyl-[protein]. The reaction is highly specific for glutamate, as demonstrated by mass spectrometry and mutagenesis studies.
Post-Modification Effects
In simple terms: The modified protein changes its behavior.
ADP-ribosylation can alter protein-protein interactions, enzymatic activity, or stability. For example, ADP-ribosylation of PARP7 and AHR marks them for degradation via the ubiquitin-proteasome system. In antiviral defense, mRNA ADP-ribosylation blocks translation, thereby inhibiting viral replication.
Reversal and Regulation
In simple terms: The modification can be removed or controlled.
The ADP-ribosylation is reversible; enzymes such as ADP-ribosylhydrolases remove the modification. Regulation also occurs at the level of enzyme expression, dimerization, and post-translational modifications. For instance, PARP15 ART domain dimerization is critical for its activity, and TIPARP mono-ADP-ribosyltransferase activity is induced by TCDD.
Key Genes Involved in GO:0140807 NAD+-protein-glutamate ADP-ribosyltransferase activity
The following genes encode enzymes with NAD+-protein-glutamate ADP-ribosyltransferase activity or are directly involved in its regulation and downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PARP1 | Poly-ADP-ribosylation; contains ADP-ribosyltransferase activity | DNA repair, cancer therapy target |
| PARP2 | Poly-ADP-ribosylation | DNA damage response |
| PARP7 | Mono-ADP-ribosylation of proteins including AHR | Cancer, immune signaling, degradation |
| PARP10 | Mono-ADP-ribosylation of histones and auto-modification | Transcription, cell cycle |
| PARP15 | Mono-ADP-ribosylation; ART domain dimerization | Regulation of activity, cancer |
| TIPARP | Mono-ADP-ribosylation; induced by TCDD | Xenobiotic response, antiviral |
| ARTD1 | ADP-ribosyltransferase | DNA repair, cell death |
| ARTD2 | ADP-ribosyltransferase | DNA repair |
| ARTD3 | ADP-ribosyltransferase | DNA repair, cancer |
| ARTD4 | ADP-ribosyltransferase | DNA repair |
| ARTD5 | ADP-ribosyltransferase | DNA repair, cancer |
| ARTD6 | ADP-ribosyltransferase | DNA repair |
| ARTD7 | Mono-ADP-ribosylation | Immune response |
| ARTD8 | Mono-ADP-ribosylation | Immune response |
| ARTD9 | Mono-ADP-ribosylation | Immune response |
| ARTD10 | Mono-ADP-ribosylation | Transcription, cell cycle |
| ARTD14 | Mono-ADP-ribosylation | Regulation of activity |
| ARTD15 | Mono-ADP-ribosylation | Regulation of activity |
How Is NAD+-protein-glutamate ADP-ribosyltransferase activity Regulated?
The activity of NAD+-protein-glutamate ADP-ribosyltransferases is regulated at multiple levels. Enzyme expression can be induced by external stimuli, such as TCDD for TIPARP. Post-translational modifications, including auto-ADP-ribosylation, can modulate activity. Dimerization of the ART domain, as shown for PARP15, is a key regulatory mechanism. Additionally, the availability of NAD+ and the presence of hydrolases that reverse the modification influence the overall levels of ADP-ribosylation.
NAD+-protein-glutamate ADP-ribosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PARP7 | Cancer, immune evasion | Knockout and overexpression in cancer cell lines |
| PARP15 | Cancer, regulation of activity | Point mutations in ART domain to study dimerization |
| TIPARP | Xenobiotic response, antiviral | Knockout in hepatocytes, TCDD treatment |
| Pertussis toxin | Whooping cough | Inhibitor screening in cell-based assays |
| C. difficile toxin | Colitis | Toxin treatment in intestinal organoids |
Cancer
ADP-ribosyltransferases such as PARP7 and PARP15 are implicated in cancer. PARP7 mono-ADP-ribosylates AHR, marking it for degradation, and its inhibition can enhance antitumor immunity. PARP15 ART domain dimerization regulates its activity, and dysregulation may contribute to cancer progression. PARP10 auto- and histone MARylation is linked to transcription and cell cycle regulation, with potential roles in cancer.
Infectious Diseases
Bacterial toxins like Clostridium difficile toxin A and pertussis toxin use ADP-ribosyltransferase activity to modify host proteins, leading to disease symptoms. Inhibitors of pertussis toxin ADP-ribosyltransferase activity are being developed as anti-virulence agents.
Antiviral Defense
An mRNA ADP-ribosyltransferase blocks translation of viral RNA, providing a defense mechanism against viruses. This highlights the role of ADP-ribosylation in innate immunity.
From NAD+-protein-glutamate ADP-ribosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the role of PARP7 in tumor immunity? | PARP7 knockout and overexpression in syngeneic mouse tumor models |
| How does PARP15 dimerization affect activity? | Point mutations disrupting ART domain dimerization |
| Does TIPARP ADP-ribosylate specific targets? | Knock-in of tagged TIPARP for proteomics |
| Can pertussis toxin activity be inhibited? | Overexpression of pertussis toxin in mammalian cells with inhibitor treatment |
| What is the impact of PARP10 on transcription? | Knockout and overexpression in cell lines, RNA-seq |
| How does mRNA ADP-ribosylation block translation? | In vitro translation assays with modified mRNA |
How to Study the NAD+-protein-glutamate ADP-ribosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Acid-urea gel electrophoresis | ADP-ribosylation status of proteins | Detection of MARylation |
| 32P-NAD+ labeling | Enzymatic activity | In vitro activity assays |
| Mass spectrometry | ADP-ribosylated residues and sites | Mapping modification sites |
| Western blot with anti-ADP-ribose | Levels of ADP-ribosylated proteins | Cell signaling studies |
| CRISPR knockout | Loss-of-function phenotypes | Gene function studies |
| CRISPR knock-in | Tagged protein expression | Proteomics and imaging |
| RNA-seq | Transcriptional changes | Pathway analysis |
| Proteomics | Protein interactions and modifications | Systems-level analysis |
Acid-Urea Gel Electrophoresis
This method separates proteins based on ADP-ribosylation status, allowing visualization of mono- and poly-ADP-ribosylated proteins. It has been used to study PARP10 auto- and histone MARylation.
Radiolabeling with 32P-NAD+
Incubating enzymes with 32P-NAD+ followed by SDS-PAGE and autoradiography detects ADP-ribosylated proteins. This approach is useful for measuring enzymatic activity in vitro.
Mass Spectrometry
Mass spectrometry identifies specific ADP-ribosylated residues, such as glutamate, and can map modification sites on target proteins. It has been used to confirm glutamate-specific ADP-ribosylation.
CRISPR-Cas9 Genome Editing
Knockout, knock-in, and point mutation models enable functional studies of ADP-ribosyltransferases in cells and organisms. For example, PARP7 knockout mice have been used to study immune responses.
How CRISPR Can Be Used to Study GO:0140807 NAD+-protein-glutamate ADP-ribosyltransferase activity
Knockout
CRISPR knockout of ADP-ribosyltransferase genes, such as PARP7 or PARP15, allows researchers to study loss-of-function phenotypes, including changes in protein stability, immune signaling, and cell survival.
Point Mutation
Introducing point mutations in the catalytic glutamate or NAD+ binding residues can abolish enzymatic activity, enabling precise structure-function studies. For example, mutations in the ART domain of PARP15 disrupt dimerization and activity.
Knock-in
Knock-in of epitope tags or fluorescent proteins allows visualization and purification of ADP-ribosyltransferases. Tagged TIPARP has been used to study its induction and interactions.
Overexpression
Overexpression of wild-type or mutant enzymes in cell lines can amplify ADP-ribosylation signals for detection and can model gain-of-function effects observed in disease.
How EDITGENE Supports NAD+-protein-glutamate ADP-ribosyltransferase activity Research
Researchers studying NAD+-protein-glutamate ADP-ribosyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in a specific cellular process or disease. This requires precise genetic models that can knockout, mutate, tag, or overexpress the gene of interest.
Contact EDITGENE today to design your custom CRISPR model for NAD+-protein-glutamate ADP-ribosyltransferase activity research.
Frequently Asked Questions About NAD+-protein-glutamate ADP-ribosyltransferase activity
What is NAD+-protein-glutamate ADP-ribosyltransferase activity?
It is an enzymatic activity that transfers ADP-ribose from NAD+ to a glutamate residue on a target protein, releasing nicotinamide.
What genes are involved in NAD+-protein-glutamate ADP-ribosyltransferase activity?
Genes include PARP1, PARP2, PARP7, PARP10, PARP15, TIPARP, and other ARTD family members.
What is the GO ID for NAD+-protein-glutamate ADP-ribosyltransferase activity?
GO:0140807.
How is glutamate-specific ADP-ribosylation detected?
Methods include acid-urea gel electrophoresis, radiolabeling with 32P-NAD+, and mass spectrometry.
What diseases are associated with ADP-ribosyltransferase activity?
Cancer, infectious diseases, and antiviral responses are linked to this activity.
Can CRISPR be used to study ADP-ribosyltransferases?
Yes, CRISPR knockout, knock-in, and point mutation models are widely used to study gene function.
What is the difference between mono- and poly-ADP-ribosylation?
Mono-ADP-ribosylation adds a single ADP-ribose unit, while poly-ADP-ribosylation adds chains; both can occur on glutamate residues.
Which bacterial toxins have this activity?
Clostridium difficile toxin A and pertussis toxin are examples.
How is PARP15 activity regulated?
Dimerization of the ART domain regulates PARP15 catalytic activity.
What is the role of TIPARP in ADP-ribosylation?
TIPARP is a mono-ADP-ribosyltransferase induced by TCDD and involved in xenobiotic response.
Conclusion
NAD+-protein-glutamate ADP-ribosyltransferase activity (GO:0140807) is a fundamental enzymatic function with broad implications in cell biology and disease. Understanding its mechanisms, regulation, and targets is essential for developing therapeutics. CRISPR-based models and advanced detection methods continue to illuminate this dynamic modification.
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. García-Saura AG et al.. 2021. PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.. Cells 10(3) PMID: 33804157
- 7. 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
- 8. 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
