GO:0140805 NAD+-protein-serine ADP-ribosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140805 describes the enzymatic transfer of an ADP-ribose moiety from NAD+ to a serine residue on a target protein, releasing nicotinamide and forming O-(ADP-D-ribosyl)-L-serine.
• This activity is carried out by ADP-ribosyltransferase (ART) enzymes, including bacterial toxins such as Clostridium difficile toxins and pertussis toxin, as well as human PARP family members like PARP7, PARP10, and PARP15.
• Serine ADP-ribosylation is a reversible post-translational modification that can alter protein function, stability, and interactions, and is emerging as a key regulator of immune signaling, transcription, and antiviral defense.
• Dysregulation of serine ADP-ribosylation is linked to cancer, infectious diseases, and inflammatory disorders, making it a promising target for therapeutic intervention.
• CRISPR-based knockout, point-mutation, and knock-in models enable precise dissection of the physiological roles of specific ART enzymes and their target serine residues.
• Advanced methods such as acid-urea gel electrophoresis, mass spectrometry, and NAD+ analog biocatalysis are used to detect and quantify serine ADP-ribosylation in cells and tissues.
Description
NAD+-protein-serine ADP-ribosyltransferase activity (GO:0140805) is a molecular function that catalyzes the transfer of an ADP-ribose group from nicotinamide adenine dinucleotide (NAD+) to the hydroxyl group of a serine residue on a protein substrate, producing nicotinamide and O-(ADP-D-ribosyl)-L-serine. This reaction is a form of mono-ADP-ribosylation and is distinct from poly-ADP-ribosylation, which typically modifies glutamate, aspartate, or lysine residues. The modification is reversible and can modulate protein function, localization, and stability, thereby influencing diverse cellular processes. Researchers study this activity because it plays critical roles in bacterial pathogenesis, host immune responses, and cancer biology. For example, Clostridium difficile toxins use this activity to disrupt host cell signaling, while human PARP enzymes such as PARP7 and PARP15 regulate interferon signaling and stress responses. Understanding the molecular mechanisms and regulatory networks of serine ADP-ribosylation is essential for developing targeted therapies against infectious diseases and cancer.
NAD+-protein-serine ADP-ribosyltransferase activity At A Glance
| GO ID | GO:0140805 |
|---|---|
| GO term | NAD+-protein-serine ADP-ribosyltransferase activity |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Transfer of ADP-ribose from NAD+ to serine residues on proteins, forming O-(ADP-D-ribosyl)-L-serine and releasing nicotinamide. |
| Reaction | L-seryl-[protein] + NAD+ = H+ + nicotinamide + O-(ADP-D-ribosyl)-L-seryl-[protein]. |
| Enzyme class | ADP-ribosyltransferase (ART) domain-containing enzymes, including bacterial toxins and human PARP family members. |
| Subcellular location | Cytoplasm, nucleus, and extracellular space depending on the enzyme and context. |
| Related diseases | Infectious diseases (e.g., C. difficile, pertussis), cancer, and inflammatory disorders. |
What Is GO:0140805?
GO:0140805, NAD+-protein-serine ADP-ribosyltransferase activity, is defined as the catalysis of the reaction: L-seryl-[protein] + NAD+ = H+ + nicotinamide + O-(ADP-D-ribosyl)-L-seryl-[protein]. In simpler terms, it is an enzymatic activity that attaches a single ADP-ribose unit to a serine residue on a target protein, using NAD+ as the donor and releasing nicotinamide as a byproduct. This activity is a type of mono-ADP-ribosylation and is mediated by enzymes containing an ADP-ribosyltransferase (ART) domain.
Why Is NAD+-protein-serine ADP-ribosyltransferase activity Important in Cell Biology?
Serine ADP-ribosylation is a rapidly expanding area of research because it represents a unique post-translational modification that regulates protein function in health and disease. Unlike other ADP-ribosylation types, serine modification is specifically catalyzed by a subset of ART enzymes and has been implicated in immune signaling, DNA repair, and antiviral defense. The activity is hijacked by bacterial toxins to cause disease, and its dysregulation in human cells contributes to cancer and inflammatory conditions. Therefore, understanding GO:0140805 provides insights into fundamental cell biology and offers opportunities for therapeutic intervention.
• Mediates host-pathogen interactions: bacterial toxins such as C. difficile toxin A and B and pertussis toxin use serine ADP-ribosyltransferase activity to modify host proteins and disrupt signaling.
• Regulates immune responses: PARP7 and other human ART enzymes modify serine residues on proteins involved in interferon signaling, affecting antiviral defense.
• Contributes to cancer biology: PARP15 and PARP10 are implicated in cancer cell proliferation and stress responses, and their ART activity is a potential drug target.
• Modulates protein stability and interactions: serine ADP-ribosylation can alter protein-protein interactions and target proteins for degradation.
• Provides a mechanism for reversible signaling: the modification can be removed by ADP-ribosylhydrolases, making it a dynamic regulatory switch.
• Enables development of chemical probes: NAD+ analogs and inhibitors are being developed to study and inhibit serine ADP-ribosyltransferases.
• Links to DNA damage response: some ART enzymes are recruited to DNA damage sites and modify serine residues on repair proteins.
• Involved in transcriptional regulation: TIPARP (PARP7) mono-ADP-ribosylates serine residues on transcription factors and co-regulators.
• Serves as a biomarker: altered serine ADP-ribosylation patterns are observed in cancer and infectious diseases.
• Facilitates structural and mechanistic studies: dimerization of ART domains regulates activity, as shown for PARP15.
What Happens During NAD+-protein-serine ADP-ribosyltransferase activity?
Substrate recognition and binding
In simple terms: The enzyme finds and grabs its target protein and NAD+.
The ART enzyme first binds to NAD+ and a target protein containing a serine residue. The ART domain recognizes specific structural features around the serine, often within a defined consensus motif. For bacterial toxins like C. difficile toxin B, the target is a specific serine in Rho GTPases. In human PARP10, auto-modification occurs on multiple serine residues, indicating broad substrate tolerance.
Catalysis and ADP-ribose transfer
In simple terms: The enzyme cuts NAD+ and attaches the ADP-ribose part to the serine.
The catalytic reaction proceeds via an SN1-like mechanism: the nicotinamide group leaves NAD+, forming an oxocarbenium intermediate, which is then attacked by the serine hydroxyl group. This results in the formation of O-(ADP-D-ribosyl)-L-serine and release of nicotinamide and a proton. The reaction is stereospecific and requires a conserved glutamate residue in the ART domain.
Post-modification effects
In simple terms: The attached ADP-ribose changes how the target protein works.
Serine ADP-ribosylation can alter the target protein's activity, localization, 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 by an ART enzyme blocks translation, thereby inhibiting viral replication.
Reversal and regulation
In simple terms: Other enzymes can remove the ADP-ribose tag to reverse the modification.
The modification is reversible; ADP-ribosylhydrolases such as MACROD1/2 and TARG1 can remove ADP-ribose from serine residues. This reversibility allows dynamic regulation of signaling pathways. Additionally, ART domain dimerization can regulate enzymatic activity, as shown for PARP15, where dimerization is required for optimal catalysis.
Key Genes Involved in GO:0140805 NAD+-protein-serine ADP-ribosyltransferase activity
The following genes encode enzymes or proteins directly involved in NAD+-protein-serine ADP-ribosyltransferase activity, including catalytic ART domains, regulatory subunits, and target proteins.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PARP7 (TIPARP) | Mono-ADP-ribosyltransferase that modifies serine residues on target proteins, including AHR and PARP7 itself | Regulates interferon signaling and is a target for cancer immunotherapy |
| PARP10 | Mono-ADP-ribosyltransferase with auto- and histone MARylation activity | Involved in DNA damage response and cancer cell proliferation |
| PARP15 | ADP-ribosyltransferase that forms dimers for catalytic activity | Dimerization regulates activity; potential drug target in cancer |
| ARTD1 (PARP1) | Poly-ADP-ribosyltransferase with mono-ADP-ribosyltransferase activity on serine residues | DNA repair and transcription regulation |
| ARTD2 (PARP2) | ADP-ribosyltransferase involved in DNA repair | May modify serine residues in response to DNA damage |
| CdtA | Clostridium difficile toxin A enzymatic subunit | Causes host cell toxicity by modifying Rho GTPases |
| CdtB | Clostridium difficile toxin B enzymatic subunit | Modifies Rho GTPases on serine residues |
| PtxS1 | Pertussis toxin S1 subunit | ADP-ribosylates Gi/Go proteins on cysteine, but related ART activity |
| TIPARP | TCDD-inducible poly-ADP-ribose polymerase | Mono-ADP-ribosylates serine residues on transcription factors |
| AHR | Aryl hydrocarbon receptor | Target of PARP7-mediated serine ADP-ribosylation and degradation |
| MACROD1 | ADP-ribosylhydrolase | Removes ADP-ribose from serine residues |
| MACROD2 | ADP-ribosylhydrolase | Reverses serine ADP-ribosylation |
| TARG1 | ADP-ribosylhydrolase | Reverses serine ADP-ribosylation |
| RhoA | Small GTPase | Target of C. difficile toxin ADP-ribosylation |
| Rac1 | Small GTPase | Target of C. difficile toxin ADP-ribosylation |
| Cdc42 | Small GTPase | Target of C. difficile toxin ADP-ribosylation |
| Histone H3 | Chromatin protein | Substrate for PARP10-mediated MARylation |
| Histone H4 | Chromatin protein | Substrate for PARP10-mediated MARylation |
How Is NAD+-protein-serine ADP-ribosyltransferase activity Regulated?
Serine ADP-ribosyltransferase activity is regulated at multiple levels. Enzymatic activity can be controlled by dimerization of the ART domain, as shown for PARP15, where dimerization is required for efficient catalysis. Auto-ADP-ribosylation can also regulate activity, as seen with PARP10, which undergoes multi-site auto-modification. Additionally, the availability of NAD+ and the presence of ADP-ribosylhydrolases that reverse the modification provide dynamic control. In bacterial toxins, activity is regulated by proteolytic activation and disulfide bond reduction.
NAD+-protein-serine ADP-ribosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CdtA/CdtB | Clostridium difficile infection | Knockout of toxin genes in C. difficile; mouse model of colitis |
| PARP7 | Cancer immunotherapy | PARP7 knockout mice; tumor cell lines with PARP7 overexpression |
| PARP15 | Cancer | PARP15 knockout cell lines; xenograft models |
| PARP10 | Cancer, DNA damage response | PARP10 knockout cells; CRISPR knock-in of catalytic mutants |
| PtxS1 | Pertussis | Pertussis toxin knockout in B. pertussis; mouse infection model |
Infectious diseases
Clostridium difficile toxins A and B are major virulence factors in antibiotic-associated diarrhea and pseudomembranous colitis. These toxins use NAD+-protein-serine ADP-ribosyltransferase activity to modify Rho GTPases, leading to cytoskeletal disruption and cell death. Pertussis toxin, produced by Bordetella pertussis, ADP-ribosylates Gi/Go proteins, contributing to whooping cough pathogenesis. Inhibitors of these ART activities are being explored as therapeutics.
Cancer
Human PARP enzymes with serine ADP-ribosyltransferase activity, such as PARP7, PARP10, and PARP15, are implicated in cancer. PARP7 mono-ADP-ribosylates AHR and itself, marking them for degradation, and its inhibition enhances antitumor immunity. PARP15 dimerization regulates its activity, and dysregulation may contribute to cancer cell survival. PARP10 auto-modification and histone MARylation are linked to DNA damage response and proliferation.
Antiviral defense
An mRNA ADP-ribosyltransferase blocks translation as a defense mechanism against viral infection. This activity modifies serine residues on mRNA or associated proteins, inhibiting viral replication. This highlights the role of serine ADP-ribosylation in innate immunity.
From NAD+-protein-serine ADP-ribosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the role of PARP7 catalytic activity in tumor immunity? | Knockout of PARP7 in mouse tumor cells, followed by syngeneic transplantation |
| How does PARP15 dimerization affect enzymatic activity? | Point mutations disrupting the dimer interface in PARP15, expressed in HEK293 cells |
| Does serine ADP-ribosylation of AHR regulate its stability? | Knock-in of serine-to-alanine mutation in AHR, followed by degradation assays |
| What are the targets of C. difficile toxin B in host cells? | Knockout of Rho GTPases in human cells, treated with toxin B |
| Can NAD+ analogs inhibit serine ADP-ribosyltransferases? | Overexpression of ART domains in E. coli, followed by in vitro assays with analogs |
| What is the impact of PARP10 auto-modification on DNA repair? | Knock-in of catalytically inactive PARP10 in cancer cells, followed by DNA damage sensitivity assays |
How to Study the NAD+-protein-serine ADP-ribosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Acid-urea gel electrophoresis | Mono-ADP-ribosylation of proteins | Detection of PARP10 auto-modification and histone MARylation |
| Mass spectrometry | Identification of ADP-ribosylated serine residues | Mapping modification sites on target proteins |
| In vitro ART assay | Enzymatic transfer of ADP-ribose from NAD+ to substrate | Screening for inhibitors or testing mutants |
| NAD+ analog biocatalysis | Incorporation of nonhydrolyzable NAD+ analogs | Studying stable ADP-ribosylation |
| CRISPR knockout screens | Genes required for ADP-ribosylation or resistance | Identifying synthetic lethal interactions |
| Western blot with anti-ADP-ribose antibodies | Global levels of ADP-ribosylation | Monitoring changes in cells treated with drugs |
| Immunoprecipitation | Protein-protein interactions of ART enzymes | Identifying target proteins |
| X-ray crystallography | Three-dimensional structure of ART domains | Understanding dimerization and catalysis |
Detection of serine ADP-ribosylation
Acid-urea gel electrophoresis is a classic method to resolve mono-ADP-ribosylated proteins from unmodified forms, as demonstrated for PARP10 auto- and histone MARylation. Mass spectrometry can identify specific serine residues modified by ADP-ribose, using enrichment with ADP-ribose-binding domains.
Enzymatic assays
In vitro ADP-ribosyltransferase assays use recombinant ART domains, NAD+, and substrate proteins. The reaction can be monitored by detecting nicotinamide release or by incorporating radiolabeled or fluorescent NAD+ analogs. High-throughput screens have identified inhibitors of pertussis toxin ART activity.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes required for serine ADP-ribosylation or for resistance to ART inhibitors. For example, knockout of PARP7 in cancer cells followed by interferon treatment reveals its role in immune evasion.
Structural and biochemical studies
X-ray crystallography and cryo-EM have revealed the structure of ART domains and their dimerization interfaces, as shown for PARP15. These studies guide the design of specific inhibitors and point mutants.
How CRISPR Can Be Used to Study GO:0140805 NAD+-protein-serine ADP-ribosyltransferase activity
Knockout
CRISPR knockout of ART genes such as PARP7, PARP10, or PARP15 eliminates their serine ADP-ribosyltransferase activity, allowing researchers to study loss-of-function phenotypes. For example, PARP7 knockout in cancer cells enhances interferon signaling and reduces tumor growth. Knockout of bacterial toxin genes in C. difficile abolishes virulence in animal models.
Point Mutation
Point mutations in the catalytic domain of ART enzymes (e.g., glutamate to alanine) abolish enzymatic activity while preserving protein structure. Such mutants are used to distinguish catalytic activity from scaffolding functions. For PARP15, point mutations disrupting the dimer interface reveal the importance of dimerization for activity.
Knock-in
Knock-in of serine-to-alanine mutations in target proteins prevents their ADP-ribosylation, enabling the study of site-specific modification. For example, knock-in of AHR serine mutants can test whether ADP-ribosylation regulates AHR stability. Knock-in of tagged ART enzymes (e.g., GFP or HA) allows visualization and immunoprecipitation.
Overexpression
Overexpression of wild-type or mutant ART enzymes in cells or bacteria is used to produce recombinant protein for in vitro assays and to study gain-of-function effects. For instance, overexpression of PARP10 in HEK293 cells followed by acid-urea gel analysis reveals auto-modification patterns. Overexpression of C. difficile toxins in E. coli is used to purify active toxin for cellular studies.
How EDITGENE Supports NAD+-protein-serine ADP-ribosyltransferase activity Research
Researchers studying NAD+-protein-serine ADP-ribosyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in a specific biological process or disease. This requires precise genetic models that can knockout, mutate, or tag the gene of interest. EDITGENE provides a comprehensive suite of CRISPR services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for NAD+-protein-serine ADP-ribosyltransferase activity research.
Frequently Asked Questions About NAD+-protein-serine ADP-ribosyltransferase activity
What is NAD+-protein-serine ADP-ribosyltransferase activity?
It is an enzymatic activity (GO:0140805) that transfers an ADP-ribose group from NAD+ to a serine residue on a protein, forming O-(ADP-D-ribosyl)-L-serine and releasing nicotinamide.
What genes are involved in NAD+-protein-serine ADP-ribosyltransferase activity?
Key genes include PARP7, PARP10, PARP15, and bacterial toxin genes such as CdtA/CdtB from C. difficile and PtxS1 from B. pertussis.
What is the difference between mono- and poly-ADP-ribosylation?
Mono-ADP-ribosylation adds a single ADP-ribose unit, often to serine, while poly-ADP-ribosylation adds chains of ADP-ribose, typically to glutamate, aspartate, or lysine residues.
How is serine ADP-ribosylation detected?
It can be detected by acid-urea gel electrophoresis, mass spectrometry, or western blot with anti-ADP-ribose antibodies.
Which diseases are linked to serine ADP-ribosylation?
It is linked to infectious diseases (e.g., C. difficile, pertussis), cancer, and inflammatory disorders.
Can serine ADP-ribosylation be reversed?
Yes, ADP-ribosylhydrolases such as MACROD1, MACROD2, and TARG1 remove ADP-ribose from serine residues.
What is the role of PARP7 in immunity?
PARP7 mono-ADP-ribosylates AHR and itself, marking them for degradation, and its inhibition enhances antitumor immunity.
How does PARP15 activity get regulated?
PARP15 activity is regulated by dimerization of its ART domain, which is required for efficient catalysis.
What experimental models are used to study serine ADP-ribosylation?
Common models include CRISPR knockout cell lines, point mutant knock-ins, and overexpression systems, as well as in vitro enzymatic assays.
What is the reaction catalyzed by GO:0140805?
L-seryl-[protein] + NAD+ = H+ + nicotinamide + O-(ADP-D-ribosyl)-L-seryl-[protein].
Conclusion
NAD+-protein-serine ADP-ribosyltransferase activity (GO:0140805) is a critical enzymatic function that regulates protein function through reversible mono-ADP-ribosylation of serine residues. It plays central roles in bacterial pathogenesis, immune signaling, and cancer, making it a high-priority target for therapeutic development. Advances in CRISPR-based models and detection methods are accelerating our understanding of this modification. EDITGENE offers comprehensive services to support research on this important activity.
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
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- 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
