GO:0031543 peptidyl-proline dioxygenase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031543 peptidyl-proline dioxygenase activity catalyzes the hydroxylation of peptidyl proline using 2-oxoglutarate, O2, and ascorbate.
• The reaction produces hydroxyproline, succinate, and CO2, and is essential for collagen maturation and oxygen sensing.
• Key enzymes include P4HA1, P4HA2, P4HA3, P4HB, and P4HTM, which are involved in collagen synthesis and hypoxia signaling.
• Dysregulation is linked to fibrosis, cancer, and connective tissue disorders.
• Research methods include hydroxylation assays, CRISPR knockout, and proteomics.
• EDITGENE provides CRISPR services to model gene function and disease mechanisms related to this activity.
Description
GO:0031543 peptidyl-proline dioxygenase activity is a molecular function that catalyzes the hydroxylation of proline residues in peptides, a modification critical for protein stability and function. This activity requires 2-oxoglutarate, molecular oxygen, and ascorbate as cofactors, and it produces hydroxyproline, succinate, and carbon dioxide. It is best known for its role in collagen biosynthesis, where hydroxyproline is essential for the triple-helical structure of collagen. Beyond collagen, this activity is involved in oxygen sensing and gene regulation. Researchers study this term to understand connective tissue diseases, cancer progression, and hypoxia responses.
peptidyl-proline dioxygenase activity At A Glance
| GO ID | GO:0031543 |
|---|---|
| GO term | peptidyl-proline dioxygenase activity |
| Ontology | molecular_function |
| Synonym | prolyl 4-hydroxylase activity |
| Major function | Hydroxylation of proline residues in peptides |
| Cofactors | 2-oxoglutarate, O2, ascorbate, Fe2+ |
| Reaction products | Hydroxyproline, succinate, CO2 |
| EC number | 1.14.11.2 |
What Is GO:0031543?
Peptidyl-proline dioxygenase activity (GO:0031543) is defined as the catalysis of the reaction: peptidyl L-proline + 2-oxoglutarate + O2 = peptidyl hydroxy-L-proline + succinate + CO2. This enzymatic activity hydroxylates proline residues in peptides, a post-translational modification that requires iron and ascorbate.
Why Is peptidyl-proline dioxygenase activity Important in Cell Biology?
Peptidyl-proline dioxygenase activity is crucial for the structural integrity of collagen and other proteins with collagen-like domains, and it plays a key role in cellular responses to hypoxia. Its dysfunction is implicated in a range of diseases, including fibrosis, cancer, and connective tissue disorders, making it a target for therapeutic intervention.
• Essential for collagen synthesis and extracellular matrix stability.
• Regulates oxygen sensing through hydroxylation of hypoxia-inducible factor (HIF).
• Involved in cancer progression and metastasis.
• Linked to fibrotic diseases such as liver and lung fibrosis.
• Plays a role in developmental processes in plants and animals.
• Target for drugs modulating collagen deposition and angiogenesis.
• Its activity varies diurnally in different tissues.
• Can be studied using CRISPR knockout models to assess gene function.
What Happens During peptidyl-proline dioxygenase activity?
Substrate Binding and Recognition
In simple terms: The enzyme grabs onto a proline-containing peptide and prepares it for modification.
The enzyme binds to peptidyl proline residues within target proteins, often in collagen-like sequences. This binding is facilitated by the enzyme's active site, which coordinates with the substrate and cofactors.
Catalytic Hydroxylation
In simple terms: The enzyme adds an oxygen atom to proline, turning it into hydroxyproline.
Using 2-oxoglutarate and molecular oxygen, the enzyme hydroxylates the proline residue, producing hydroxyproline, succinate, and CO2. This reaction requires iron and ascorbate to maintain the enzyme's activity.
Product Release and Recycling
In simple terms: The modified protein is released, and the enzyme is ready for another round.
After hydroxylation, the hydroxyproline-containing peptide is released. The enzyme can then bind new substrates, continuing the cycle. Ascorbate is consumed and must be regenerated to sustain activity.
Key Genes Involved in GO:0031543 peptidyl-proline dioxygenase activity
The following genes encode enzymes with peptidyl-proline dioxygenase activity or are directly involved in its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| P4HA1 | Catalytic subunit of prolyl 4-hydroxylase | Collagen synthesis, cancer |
| P4HA2 | Catalytic subunit of prolyl 4-hydroxylase | Collagen synthesis, hypoxia |
| P4HA3 | Catalytic subunit of prolyl 4-hydroxylase | Collagen synthesis, cancer |
| P4HB | Protein disulfide isomerase, beta subunit | Collagen folding, ER stress |
| P4HTM | Transmembrane prolyl 4-hydroxylase | Oxygen sensing, HIF regulation |
| EGLN1 | Prolyl hydroxylase domain protein 2 | Hypoxia signaling |
| EGLN2 | Prolyl hydroxylase domain protein 1 | Hypoxia signaling |
| EGLN3 | Prolyl hydroxylase domain protein 3 | Hypoxia signaling |
| HIF1A | Hypoxia-inducible factor 1-alpha | Substrate for hydroxylation |
| COL1A1 | Collagen type I alpha 1 chain | Substrate for hydroxylation |
| COL1A2 | Collagen type I alpha 2 chain | Substrate for hydroxylation |
| COL3A1 | Collagen type III alpha 1 chain | Substrate for hydroxylation |
| VHL | von Hippel-Lindau tumor suppressor | Recognizes hydroxyproline |
| EPAS1 | Endothelial PAS domain protein 1 | Hypoxia signaling |
| ARNT | Aryl hydrocarbon receptor nuclear translocator | Hypoxia signaling |
| CREBBP | CREB binding protein | Transcriptional coactivator |
| EP300 | E1A binding protein p300 | Transcriptional coactivator |
How Is peptidyl-proline dioxygenase activity Regulated?
Peptidyl-proline dioxygenase activity is regulated by oxygen availability, iron and ascorbate levels, and 2-oxoglutarate concentration. The activity of prolyl hydroxylases is also modulated by post-translational modifications and interactions with other proteins. In hypoxia, reduced oxygen levels inhibit the activity, leading to stabilization of HIF and activation of hypoxia-responsive genes.
peptidyl-proline dioxygenase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| P4HA1 | Fibrosis, cancer | Knockout mice, overexpression cell lines |
| P4HA2 | Hypoxia, cancer | CRISPR knockout in cancer cells |
| P4HTM | Obesity, cognitive impairment | Knockout mice |
| EGLN1 | Hypoxia, polycythemia | Point mutation knock-in |
| COL1A1 | Osteogenesis imperfecta | Knock-in of patient mutations |
Fibrosis and Connective Tissue Disorders
Dysregulated peptidyl-proline dioxygenase activity leads to excessive collagen deposition, contributing to fibrosis in liver, lung, and kidney. Mutations in genes encoding these enzymes can cause connective tissue disorders such as Ehlers-Danlos syndrome.
Cancer
Increased prolyl hydroxylase activity is associated with tumor progression and metastasis, as it promotes collagen remodeling and angiogenesis. Inhibitors are being explored as anticancer agents.
Hypoxia and Ischemia
Prolyl hydroxylases act as oxygen sensors; their inhibition under hypoxia stabilizes HIF, affecting angiogenesis and metabolism. This pathway is critical in ischemic diseases and cancer.
From peptidyl-proline dioxygenase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Enzyme catalytic mechanism | Recombinant protein, point mutations |
| Role in collagen synthesis | Knockout fibroblasts |
| Hypoxia signaling | Knockout cancer cells |
| Disease-associated mutations | Knock-in mice |
| Enzyme localization | Tagged knock-in |
| Overexpression effects | Stable cell lines |
How to Study the peptidyl-proline dioxygenase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Hydroxylation assay | Enzyme activity | Kinetic studies, inhibitor screening |
| CRISPR knockout | Gene function | Target validation |
| Mass spectrometry | Hydroxyproline sites | Substrate identification |
| Western blot | HIF levels | Hypoxia response |
| Reporter assay | Transcriptional activity | Hypoxia signaling |
| Immunohistochemistry | Protein localization | Tissue distribution |
| RNA-seq | Gene expression changes | Pathway analysis |
Enzymatic Activity Assays
Hydroxylation activity can be measured using radiolabeled proline or by detecting succinate production. These assays are used to screen inhibitors and study kinetics.
CRISPR-Cas9 Knockout
Knockout of genes encoding prolyl hydroxylases allows assessment of their role in collagen synthesis and hypoxia signaling. This method is useful for validating drug targets.
Proteomics and Mass Spectrometry
Mass spectrometry identifies hydroxyproline sites in proteins, providing insights into substrate specificity and modification patterns.
Hypoxia Response Assays
HIF stabilization and transcriptional activity can be measured using reporter assays and Western blotting.
How CRISPR Can Be Used to Study GO:0031543 peptidyl-proline dioxygenase activity
Knockout
CRISPR knockout of P4HA1, P4HA2, or P4HTM can abolish peptidyl-proline dioxygenase activity, leading to defective collagen synthesis and altered hypoxia responses.
Point Mutation
Introducing point mutations in the catalytic domain can dissect the enzymatic mechanism and identify residues critical for substrate binding or catalysis.
Knock-in
Knock-in of disease-associated mutations or tags allows study of protein function in a physiological context.
Overexpression
Overexpression of prolyl hydroxylases can enhance collagen deposition or suppress HIF signaling, useful for studying fibrosis and cancer.
How EDITGENE Supports peptidyl-proline dioxygenase activity Research
Researchers studying peptidyl-proline dioxygenase activity-related genes often need to determine whether a candidate gene is causally involved in collagen synthesis, hypoxia signaling, or disease progression. EDITGENE provides comprehensive CRISPR services to create precise cellular and animal models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for peptidyl-proline dioxygenase activity research.
Frequently Asked Questions About peptidyl-proline dioxygenase activity
What is peptidyl-proline dioxygenase activity?
It is an enzymatic activity that hydroxylates proline residues in peptides, using 2-oxoglutarate, oxygen, and ascorbate.
What genes are involved in peptidyl-proline dioxygenase activity?
Key genes include P4HA1, P4HA2, P4HA3, P4HB, and P4HTM.
What diseases are associated with peptidyl-proline dioxygenase activity?
Fibrosis, cancer, and connective tissue disorders.
How is peptidyl-proline dioxygenase activity regulated?
By oxygen levels, iron, ascorbate, and 2-oxoglutarate.
What is the role of prolyl hydroxylase in collagen synthesis?
It hydroxylates proline in collagen, essential for triple helix stability.
Can CRISPR be used to study peptidyl-proline dioxygenase activity?
Yes, knockout and knock-in models are valuable.
What are the cofactors for peptidyl-proline dioxygenase?
Iron, ascorbate, 2-oxoglutarate, and oxygen.
How to measure peptidyl-proline dioxygenase activity?
Using hydroxylation assays or mass spectrometry.
What is the GO ID for peptidyl-proline dioxygenase activity?
GO:0031543.
What are synonyms for peptidyl-proline dioxygenase activity?
Prolyl 4-hydroxylase activity, proline hydroxylase activity.
Conclusion
Peptidyl-proline dioxygenase activity is a fundamental enzymatic function with critical roles in collagen biology and oxygen sensing. Its dysregulation contributes to major diseases, making it a prime target for therapeutic development. EDITGENE offers advanced CRISPR solutions to study this activity and accelerate drug discovery.
References
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- 3. Myllylä R et al.. 1989. The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity.. Biochem J 263(2):609-11 PMID: 2557001
- 4. Schnicker NJ et al.. 2016. Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns.. J Biol Chem 291(25):13360-74 PMID: 27129244
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- 6. de Jong L et al.. 1982. Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation.. Biochim Biophys Acta 704(2):326-32 PMID: 6285984
- 7. Counts DF et al.. 1978. Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.. Proc Natl Acad Sci U S A 75(5):2145-9 PMID: 209453
- 8. Myllykoski M et al.. 2021. Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains.. J Biol Chem 296:100197 PMID: 33334883
