GO:0140802 NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0140802 describes the enzymatic transfer of an ADP-ribosyl group from NAD+ to the C-terminal glycine of a target protein, releasing nicotinamide.
• This activity is a molecular_function that modifies protein C-termini, a post-translational modification distinct from poly-ADP-ribosylation on internal residues.
• The reaction is catalyzed by ADP-ribosyl transferases, including bacterial effectors such as AvrRpm1 that modify host proteins to promote virulence.
• C-terminal glycine tagging is a widely used recombinant protein engineering strategy, and its unintended ADP-ribosylation can affect protein behavior.
• Dysregulation of ADP-ribosylation is linked to cancer, neurodegeneration, and infectious diseases, making this activity a therapeutic target.
• CRISPR-based knockout, knock-in, and point-mutation models enable precise dissection of this enzymatic activity in disease contexts.
Description
NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity (GO:0140802) is a molecular function that catalyzes the transfer of an ADP-ribosyl moiety from NAD+ to the C-terminal glycine residue of a target protein, producing a C-terminal O-(ADP-D-ribosyl)-glycine modification and nicotinamide. This activity represents a specialized form of ADP-ribosylation, a reversible post-translational modification that regulates protein function, stability, and interactions in diverse cellular processes. Unlike poly-ADP-ribosylation that occurs on internal glutamate, aspartate, or serine residues, this activity specifically targets the protein C-terminus, adding a unique layer of regulatory control. Researchers study this activity to understand how bacterial pathogens manipulate host proteins, how cellular enzymes maintain proteostasis, and how dysregulation contributes to diseases such as cancer and neurodegeneration. The reaction is also relevant to biotechnology, as C-terminal glycine tags are commonly used for protein purification and detection, and unintended ADP-ribosylation of such tags can alter protein properties. Understanding GO:0140802 at the molecular level is essential for developing targeted therapies and for interpreting experimental results involving tagged proteins.
NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity At A Glance
| GO ID | GO:0140802 |
|---|---|
| GO term | NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Transfer of ADP-ribose from NAD+ to the C-terminal glycine of a protein, releasing nicotinamide |
| Reaction | [protein]-C-terminal glycine + NAD+ = [protein]-C-terminal O-(ADP-D-ribosyl)-glycine + nicotinamide |
| Substrates | NAD+ and a protein with a C-terminal glycine |
| Products | ADP-ribosylated protein and nicotinamide |
| Related activity | ADP-ribosyltransferase activity; protein ADP-ribosylation |
What Is GO:0140802?
GO:0140802 is defined as the catalysis of the reaction: [protein]-C-terminal glycine + NAD+ = [protein]-C-terminal O-(ADP-D-ribosyl)-glycine + nicotinamide. In other words, it is an enzymatic activity that attaches an ADP-ribose unit to the terminal glycine of a protein, using NAD+ as the donor and releasing nicotinamide as a byproduct.
Why Is NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity Important in Cell Biology?
GO:0140802 is important because it represents a specific enzymatic activity that modifies protein C-termini, a modification that can alter protein stability, localization, and interactions. This activity is exploited by bacterial pathogens such as AvrRpm1 to modify host proteins and promote virulence. In human cells, ADP-ribosylation regulates key processes including DNA repair, cell cycle, and apoptosis, and its dysregulation is implicated in cancer and neurodegeneration. Understanding this activity also aids in interpreting experiments that use C-terminal glycine tags, as unintended ADP-ribosylation can confound results.
• Modifies protein C-termini, affecting protein function and stability.
• Plays a role in host-pathogen interactions, as bacterial effectors like AvrRpm1 use this activity to modify host proteins.
• Contributes to the regulation of cell death pathways, including apoptosis, through modification of proteins such as HIPK2.
• Linked to cancer biology, as ADP-ribosylation regulates proteins involved in proliferation and survival.
• Implicated in neurodegeneration, with SIRT2 and GARS mutations affecting neuropathy.
• Relevant to biotechnology, as C-terminal glycine tags are common in recombinant proteins and can be modified.
• Provides a mechanism for reversible post-translational regulation, influencing signaling networks.
• Serves as a potential therapeutic target for infectious diseases and cancer.
• Enables precise protein engineering through CRISPR knock-in of C-terminal tags.
• Helps explain off-target effects in tagged protein experiments.
What Happens During NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity?
Substrate recognition and binding
In simple terms: The enzyme finds and grabs its two starting materials: NAD+ and a protein that ends with glycine.
The first step in the reaction is the binding of the enzyme to its substrates: NAD+ and a target protein bearing a C-terminal glycine. This recognition is mediated by the enzyme's active site, which positions the NAD+ molecule and the terminal glycine in close proximity for catalysis. The specificity for C-terminal glycine distinguishes this activity from other ADP-ribosyltransferases that modify internal residues.
Catalysis and ADP-ribose transfer
In simple terms: The enzyme cuts NAD+ and attaches the ADP-ribose part onto the end of the protein.
Upon binding, the enzyme catalyzes the cleavage of the glycosidic bond in NAD+, releasing nicotinamide and forming an ADP-ribose-enzyme intermediate or directly transferring the ADP-ribose to the C-terminal glycine of the target protein. This results in the formation of a C-terminal O-(ADP-D-ribosyl)-glycine modification. The reaction is reversible in principle, but the reverse reaction is not favored under physiological conditions.
Product release and modification consequences
In simple terms: The modified protein is released and the new tag can change how the protein behaves.
After the transfer, the ADP-ribosylated protein and nicotinamide are released. The newly added ADP-ribose moiety can alter the protein's charge, conformation, and interaction partners, thereby modulating its function. For example, ADP-ribosylation of HIPK2 by PARP1 regulates its stability and proapoptotic function. In bacterial systems, AvrRpm1-mediated ADP-ribosylation of host proteins promotes virulence.
Reversibility and regulation
In simple terms: Other enzymes can remove the ADP-ribose tag, making the modification reversible.
The ADP-ribosylation is reversible; enzymes such as ADP-ribosyl hydrolases can remove the modification, restoring the original C-terminal glycine. This reversibility allows dynamic regulation of protein function. The balance between transferase and hydrolase activities determines the extent and duration of the modification, influencing cellular outcomes.
Key Genes Involved in GO:0140802 NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity
The following genes and proteins are experimentally linked to ADP-ribosylation processes and related pathways, providing a starting point for functional studies of GO:0140802.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PARP1 | Poly(ADP-ribose) polymerase 1; catalyzes ADP-ribosylation of target proteins | Regulates HIPK2 stability and apoptosis; widely studied in cancer |
| SIRT2 | NAD+-dependent deacetylase; involved in neuroprotection | Knockdown rescues GARS-induced Charcot-Marie-Tooth neuropathy |
| SIRT5 | NAD+-dependent desuccinylase | Regulates SHMT2 to drive cancer cell proliferation |
| GARS | Glycyl-tRNA synthetase; mutations cause neuropathy | Model for Charcot-Marie-Tooth disease; modified by SIRT2 |
| SHMT2 | Serine hydroxymethyltransferase 2; one-carbon metabolism | Desuccinylated by SIRT5; promotes cancer proliferation |
| HIPK2 | Homeodomain-interacting protein kinase 2; proapoptotic kinase | Stability regulated by PARP1-mediated ADP-ribosylation |
| AvrRpm1 | Bacterial effector with ADP-ribosyl transferase activity | Modifies host NOI domain proteins to promote virulence |
| RPM1-interacting protein4 (RIN4) | Plant immune regulator | Target of AvrRpm1 ADP-ribosylation in Arabidopsis and soybean |
| TNT | Tuberculosis necrotizing toxin; NAD+ glycohydrolase | Exhibits NAD+ and NADP+ glycohydrolase activity |
| Diphtheria toxin | Bacterial toxin; ADP-ribosylates EF-2 | Formaldehyde-induced modifications studied by mass spectrometry |
| Iga beta | Outer membrane protein of Neisseria gonorrhoeae | C-terminal glycine-histidine tagging studied for protein engineering |
| NOI domain-containing proteins | Plant proteins with NOI domains | Targets of AvrRpm1 ADP-ribosylation |
| NAD+ | Coenzyme; substrate for ADP-ribosylation | Central to the reaction; levels affect activity |
| Nicotinamide | Byproduct of ADP-ribosylation | Can be recycled to NAD+; inhibits some ADP-ribosyltransferases |
| ADP-ribosyl hydrolases | Remove ADP-ribose from proteins | Reverse the modification; regulate dynamics |
| Mitochondrial proteins | Targets of ADP-ribosylation in mitochondria | Mitochondrial dysfunction linked to cholestatic liver injury |
How Is NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity Regulated?
The activity of NAD+-protein-C-terminal glycine ADP-ribosyltransferase is regulated by the availability of NAD+, the expression and activity of the transferase enzymes, and the opposing action of ADP-ribosyl hydrolases. NAD+ levels fluctuate with cellular metabolic state, influencing the rate of ADP-ribosylation. Additionally, post-translational modifications of the transferases themselves, such as phosphorylation or acetylation, can modulate their activity. In bacterial systems, effector proteins like AvrRpm1 are regulated by host factors and secretion signals. The balance between ADP-ribosylation and de-ADP-ribosylation is critical for cellular homeostasis, and its disruption contributes to disease.
NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PARP1 | Cancer; apoptosis regulation | Knockout or point-mutation in cancer cell lines |
| SIRT2 | Charcot-Marie-Tooth neuropathy | Knockdown or knockout in neuronal cells |
| SIRT5 | Cancer proliferation | Knockout in cancer cell lines; metabolic assays |
| GARS | Charcot-Marie-Tooth neuropathy | Knock-in of patient mutations in iPSC-derived neurons |
| AvrRpm1 | Plant immunity; bacterial virulence | Overexpression in Arabidopsis or soybean |
Cancer
ADP-ribosylation regulates proteins involved in DNA repair, cell cycle, and apoptosis. PARP1-mediated ADP-ribosylation of HIPK2 affects its stability and proapoptotic function, influencing cancer cell survival. SIRT5 desuccinylates SHMT2 to drive cancer cell proliferation, linking NAD+-dependent modifications to tumor metabolism. Dysregulation of these pathways can promote oncogenesis, making ADP-ribosylation enzymes therapeutic targets.
Neurodegeneration
SIRT2 knockdown rescues GARS-induced Charcot-Marie-Tooth neuropathy, indicating that NAD+-dependent modifications are involved in peripheral neuropathies. GARS mutations cause neuropathy, and modulation of SIRT2 activity may offer therapeutic benefit. This highlights the importance of ADP-ribosylation and related NAD+ metabolism in neuronal health.
Infectious diseases
Bacterial pathogens use ADP-ribosyl transferases to modify host proteins. AvrRpm1 from Pseudomonas syringae functions as an ADP-ribosyl transferase to modify NOI domain-containing proteins, including RPM1-interacting protein4, to promote virulence in Arabidopsis and soybean. The tuberculosis necrotizing toxin (TNT) is an NAD+ glycohydrolase with distinct enzymatic properties, contributing to mycobacterial pathogenesis. Diphtheria toxin ADP-ribosylates elongation factor 2, inhibiting protein synthesis.
Liver injury
Mitochondrial dysfunction-mediated hepatocyte senescence is involved in cholestatic liver injury, and ADP-ribosylation of mitochondrial proteins may contribute to this process. NAD+ depletion and altered ADP-ribosylation are observed in cholestatic liver injury models.
From NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does the candidate gene have ADP-ribosyltransferase activity? | In vitro enzymatic assay with recombinant protein and NAD+ |
| What is the role of C-terminal glycine in protein function? | Point mutation of the terminal glycine to alanine |
| How does ADP-ribosylation affect protein stability? | Knock-in of tagged protein followed by proteasome inhibitor treatment |
| Which proteins are modified by a specific transferase? | Overexpression of transferase followed by mass spectrometry |
| Does loss of the transferase affect disease phenotypes? | Knockout in disease-relevant cell lines or animal models |
| Can the modification be reversed? | Treatment with ADP-ribosyl hydrolase or NAD+ depletion |
How to Study the NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro ADP-ribosyltransferase assay | Enzymatic transfer of ADP-ribose from NAD+ to protein | Confirming GO:0140802 activity of a candidate enzyme |
| Mass spectrometry | Identification and mapping of ADP-ribosylated residues | Detecting C-terminal glycine ADP-ribosylation in cells |
| CRISPR knockout screening | Genes required for ADP-ribosylation or cellular responses | Identifying regulators of NAD+ metabolism |
| Western blot with anti-ADP-ribose antibody | Levels of ADP-ribosylated proteins | Monitoring changes in ADP-ribosylation upon treatment |
| NAD+ quantification | Intracellular NAD+ levels | Assessing substrate availability for ADP-ribosylation |
| Fluorescence microscopy | Subcellular localization of ADP-ribosylated proteins | Visualizing modification in situ |
| Apoptosis assays | Cell death pathways affected by ADP-ribosylation | Evaluating functional consequences of HIPK2 modification |
| Site-directed mutagenesis | Effect of C-terminal glycine mutation on modification | Testing the requirement for the terminal glycine |
Enzymatic assays
In vitro ADP-ribosyltransferase assays using recombinant enzymes and NAD+ are used to measure activity. These assays can employ radiolabeled NAD+ or fluorescent NAD+ analogs to detect ADP-ribose incorporation into protein substrates. Such assays are essential for confirming that a candidate protein possesses GO:0140802 activity.
Mass spectrometry
Mass spectrometry-based proteomics identifies ADP-ribosylated proteins and maps modification sites. This approach can detect C-terminal ADP-ribosylation on glycine residues, providing direct evidence for GO:0140802 activity in cells. Formaldehyde-induced modifications in diphtheria toxin have been characterized by mass spectrometry, illustrating the utility of this method.
CRISPR screening
Genome-wide CRISPR knockout screens can identify genes required for ADP-ribosylation or for cellular responses to this modification. Such screens have been used to uncover regulators of NAD+ metabolism and ADP-ribosylation pathways. Libraries targeting ADP-ribosyltransferases and hydrolases enable systematic functional analysis.
Imaging and cellular assays
Fluorescence microscopy with ADP-ribose-specific antibodies or tagged proteins allows visualization of ADP-ribosylation in cells. Cellular assays measuring NAD+ levels, cell viability, and apoptosis provide functional readouts of GO:0140802 activity. These methods link enzymatic activity to cellular phenotypes.
How CRISPR Can Be Used to Study GO:0140802 NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity
Knockout
CRISPR knockout of genes encoding ADP-ribosyltransferases or related enzymes can abolish GO:0140802 activity, allowing researchers to study loss-of-function phenotypes. For example, knockout of PARP1 or SIRT2 has been used to investigate their roles in cancer and neurodegeneration. Knockout models are essential for determining whether a gene is required for a specific biological process.
Point Mutation
CRISPR point mutation can introduce specific amino acid changes in the catalytic domain of ADP-ribosyltransferases or in the C-terminal glycine of substrate proteins. Mutating the terminal glycine to alanine prevents ADP-ribosylation, enabling precise dissection of the modification's function. Point mutations in GARS have been modeled to study Charcot-Marie-Tooth neuropathy.
Knock-in
CRISPR knock-in can add tags, such as C-terminal glycine-histidine tags, to proteins of interest to facilitate purification and detection. However, such tags can themselves be substrates for ADP-ribosylation, potentially affecting protein behavior. Knock-in of disease-associated mutations, such as those in GARS, creates isogenic models for studying neuropathy.
Overexpression
CRISPR activation or cDNA overexpression can increase the levels of ADP-ribosyltransferases or substrate proteins, enhancing the detection of ADP-ribosylation events. Overexpression of AvrRpm1 in plant cells has been used to study its ADP-ribosyltransferase activity and virulence function. Overexpression models are useful for identifying downstream effects of enhanced GO:0140802 activity.
How EDITGENE Supports NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity Research
Researchers studying NAD+-protein-C-terminal glycine 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, tag, or overexpress the gene of interest. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate such studies, from single-gene editing to genome-wide screening.
Contact EDITGENE today to design your custom CRISPR model for NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity research.
Frequently Asked Questions About NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity
What is NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity?
It is an enzymatic activity (GO:0140802) that transfers an ADP-ribose group from NAD+ to the C-terminal glycine of a protein, releasing nicotinamide.
What genes are involved in NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity?
Genes encoding ADP-ribosyltransferases such as PARP1, bacterial effectors like AvrRpm1, and related enzymes including SIRT2 and SIRT5 are involved.
What is the reaction catalyzed by GO:0140802?
The reaction is: [protein]-C-terminal glycine + NAD+ = [protein]-C-terminal O-(ADP-D-ribosyl)-glycine + nicotinamide.
How is NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity regulated?
It is regulated by NAD+ availability, enzyme expression, and opposing ADP-ribosyl hydrolase activity.
What diseases are associated with ADP-ribosylation?
ADP-ribosylation is linked to cancer, neurodegeneration, infectious diseases, and liver injury.
How can I study GO:0140802 in the lab?
You can use in vitro enzymatic assays, mass spectrometry, CRISPR knockout or knock-in models, and imaging techniques.
What is the role of C-terminal glycine in ADP-ribosylation?
The C-terminal glycine is the specific acceptor residue for ADP-ribose transfer, and its mutation prevents the modification.
Can CRISPR be used to study ADP-ribosyltransferase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models enable precise functional studies of ADP-ribosylation.
What is the difference between ADP-ribosylation and poly-ADP-ribosylation?
ADP-ribosylation adds a single ADP-ribose unit, while poly-ADP-ribosylation adds chains; GO:0140802 specifically modifies the C-terminal glycine.
How does bacterial AvrRpm1 use ADP-ribosylation?
AvrRpm1 functions as an ADP-ribosyl transferase to modify NOI domain-containing proteins, including RPM1-interacting protein4, to promote virulence.
Conclusion
NAD+-protein-C-terminal glycine ADP-ribosyltransferase activity (GO:0140802) is a specialized enzymatic function that modifies protein C-termini, influencing protein function and cellular processes. Its roles in bacterial pathogenesis, cancer, and neurodegeneration make it a significant research target. Understanding this activity requires precise genetic models and biochemical assays, which can be efficiently generated using CRISPR technologies. Continued investigation of GO:0140802 will uncover new therapeutic opportunities and deepen our knowledge of post-translational regulation.
References
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- 4. Yang X et al.. 2018. SHMT2 Desuccinylation by SIRT5 Drives Cancer Cell Proliferation.. Cancer Res 78(2):372-386 PMID: 29180469
- 5. Redditt TJ et al.. 2019. AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI Domain-Containing Proteins, Including Arabidopsis and Soybean RPM1-Interacting Protein4.. Plant Cell 31(11):2664-2681 PMID: 31727786
- 6. Choi JR et al.. 2016. PARP1 regulates the protein stability and proapoptotic function of HIPK2.. Cell Death Dis 7(10):e2438 PMID: 27787517
- 7. Tak U et al.. 2019. The tuberculosis necrotizing toxin is an NAD(+) and NADP(+) glycohydrolase with distinct enzymatic properties.. J Biol Chem 294(9):3024-3036 PMID: 30593509
- 8. Strauss A et al.. 1995. C-terminal glycine-histidine tagging of the outer membrane protein Iga beta of Neisseria gonorrhoeae.. FEMS Microbiol Lett 127(3):249-54 PMID: 7758939
