Recombinant Mouse Prolyl 4-hydroxylase subunit alpha-3 (P4ha3)

What is P4HA3 and what is its primary function in mice?

P4HA3 (Prolyl 4-hydroxylase subunit alpha-3) is a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis composed of two identical alpha subunits and two beta subunits. The alpha subunit provides the major part of the catalytic site of the active enzyme . In collagen and related proteins, this enzyme catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains . This hydroxylation is critical for stabilizing the triple helical structure of collagen, which is fundamental to extracellular matrix integrity in various tissues.

How is mouse P4HA3 structurally different from human and rat orthologs?

While mouse P4HA3 shares significant sequence homology with its human and rat orthologs, species-specific variations exist primarily in non-catalytic regions. The functional domains remain highly conserved across species, particularly the catalytic region that facilitates hydroxylation. The rat P4HA3 sequence (residues 25-544) contains regions critical for substrate binding and catalytic activity , and these regions are similarly preserved in mouse P4HA3. When designing experiments using recombinant mouse P4HA3, researchers should be aware that while the core functional domains are conserved, species-specific differences may influence interaction with other proteins or regulatory mechanisms.

What expression systems are optimal for producing functional recombinant mouse P4HA3?

Several expression systems can be employed for recombinant mouse P4HA3 production, including E. coli, yeast, baculovirus, and mammalian cell systems . The choice depends on your experimental requirements:

• E. coli system: Offers high yield and cost-effectiveness but may lack proper post-translational modifications

• Yeast system: Provides some post-translational modifications with moderate yield

• Baculovirus system: Delivers protein with more complex modifications and better folding

• Mammalian cell system: Produces protein with the most physiologically relevant modifications

For enzymatic activity studies requiring properly folded protein with post-translational modifications, mammalian or baculovirus systems are recommended. For structural studies where high yield is prioritized over modifications, E. coli systems may be sufficient .

What are the validated methods for assessing P4HA3 enzymatic activity in vitro?

To evaluate mouse P4HA3 enzymatic activity, researchers can employ several approaches:

• Hydroxylation assay: Measure the conversion of proline to hydroxyproline in collagen peptide substrates using liquid chromatography-mass spectrometry (LC-MS)

• Oxygen consumption assay: Quantify oxygen consumption during the hydroxylation reaction using an oxygen electrode

• Coupled enzyme assay: Monitor the oxidation of 2-oxoglutarate to succinate during the hydroxylation reaction

When conducting these assays, ensure proper co-factor inclusion (Fe²⁺, 2-oxoglutarate, ascorbate) and optimal pH (typically 7.4-8.0). The functional activity of recombinant P4HA3 can be significantly affected by the expression system used, with mammalian-expressed protein generally showing higher specific activity compared to bacterial systems .

How can I verify the purity and integrity of recombinant mouse P4HA3?

To verify recombinant mouse P4HA3 quality:

• SDS-PAGE analysis: Assess purity with protein expected at approximately 61 kDa for mouse P4HA3

• Western blot: Confirm identity using specific antibodies against P4HA3 or attached epitope tags

• Mass spectrometry: Verify sequence integrity and identify potential post-translational modifications

• Size-exclusion chromatography: Evaluate aggregation state and homogeneity

High-quality recombinant P4HA3 should demonstrate >90% purity on SDS-PAGE . When working with tagged variants, ensure the tag doesn't interfere with enzymatic activity through comparative activity assays with untagged protein.

What are appropriate storage conditions to maintain recombinant mouse P4HA3 stability?

For optimal stability of recombinant mouse P4HA3:

• Short-term storage (1-2 weeks): Store at 4°C in buffer containing 5-50% glycerol

• Long-term storage: Store at -20°C or -80°C with glycerol as cryoprotectant

• Lyophilized format: Store at -20°C with desiccant

• Working aliquots: Prepare single-use aliquots to avoid repeated freeze-thaw cycles

The recommended buffer composition typically includes Tris/PBS-based buffer (pH 7.5-8.0) with 5-50% glycerol for liquid formulations, or Tris/PBS-based buffer with 6% trehalose (pH 8.0) for lyophilized preparations . Stability studies indicate that recombinant P4HA3 retained in these conditions maintains >90% activity for at least 12 months when properly stored.

How is P4HA3 expression altered in mouse cancer models?

Similar to human cancers, mouse P4HA3 expression is significantly altered in various cancer models. Analysis of mouse tumor tissues has revealed upregulation of P4HA3 in multiple cancer types, particularly in models with extensive extracellular matrix remodeling. This overexpression pattern mirrors findings in human cancers, where P4HA3 is significantly increased in tumor tissues compared to normal tissues .

In experimental studies with mouse models, P4HA3 upregulation correlates with increased tumor stage and poor prognosis indicators. The elevated expression particularly associates with cancer progression features such as invasion and metastasis, suggesting conserved functions between human and mouse P4HA3 in cancer biology .

What approaches are effective for modulating P4HA3 activity in mouse models?

To modulate P4HA3 activity in mouse models, researchers can employ:

• Genetic approaches:

  • CRISPR/Cas9-mediated knockout or knockin models

  • Conditional knockout models using Cre-loxP systems

  • RNA interference (siRNA or shRNA) for transient knockdown

• Pharmacological approaches:

  • Prolyl hydroxylase inhibitors (with consideration for specificity)

  • Small molecules targeting P4HA3-specific regions

• Expression modulation:

  • Viral vector-mediated overexpression

  • Anti-sense oligonucleotides

When designing P4HA3 modulation studies, consider tissue-specific and temporal regulation to avoid developmental complications, as P4HA3 plays roles in normal collagen synthesis. Validation of modulation should include both mRNA and protein level assessments, as post-transcriptional regulation can significantly impact final protein levels .

What is known about the role of P4HA3 in mouse tumor immune microenvironment?

P4HA3 has emerging roles in the tumor immune microenvironment (TIME) in mice, paralleling findings in human cancer studies. Research indicates significant correlations between P4HA3 expression and immune cell infiltration patterns in mouse tumor models .

Key findings include:

• Positive correlation between P4HA3 expression and infiltration of specific immune cell populations, including macrophages and neutrophils

• Association with immunomodulatory pathways influencing T-cell function

• Impact on extracellular matrix composition affecting immune cell migration and function

Mechanistically, P4HA3-mediated collagen modifications may create physical barriers affecting immune cell infiltration and distribution within tumors. Additionally, P4HA3 activity influences hypoxic conditions in the tumor microenvironment, which can modulate immune cell function and phenotype . Understanding these interactions provides valuable insights for immunotherapy approaches in mouse cancer models.

How do post-translational modifications affect mouse P4HA3 function?

Post-translational modifications (PTMs) significantly impact mouse P4HA3 function through multiple mechanisms:

• Phosphorylation: Regulates enzymatic activity and protein-protein interactions

• Glycosylation: Influences protein stability and localization

• Ubiquitination: Controls protein turnover and degradation pathways

The selection of expression system is critical when studying PTM effects, as bacterial systems like E. coli cannot reproduce mammalian PTM patterns . For studies focusing on physiologically relevant P4HA3 activity, mammalian expression systems are preferred as they maintain the natural PTM profile.

Experimentally, site-directed mutagenesis of key modification sites combined with activity assays can elucidate the functional importance of specific PTMs. Mass spectrometry approaches enable comprehensive mapping of PTMs on recombinant mouse P4HA3, providing insights into regulatory mechanisms governing its function.

What protein interaction partners of mouse P4HA3 have been identified and characterized?

Mouse P4HA3 functions within a complex network of protein interactions. Key interaction partners include:

• P4HB (Protein disulfide isomerase): Forms the functional prolyl 4-hydroxylase tetramer

• Collagen chains: Serve as substrates for hydroxylation

• Molecular chaperones: Including HSP47, which assists in proper collagen folding

• Regulatory proteins: Including factors controlling endoplasmic reticulum stress responses

Characterization methods for these interactions include:

• Co-immunoprecipitation followed by mass spectrometry

• Yeast two-hybrid screening

• Proximity labeling approaches (BioID, APEX)

• Fluorescence resonance energy transfer (FRET)

Understanding these interaction networks provides insights into P4HA3 regulation and function within the complex cellular environment of collagen synthesis and modification .

How can mutational analysis of mouse P4HA3 inform structure-function relationships?

Mutational analysis of mouse P4HA3 provides critical insights into structure-function relationships:

• Catalytic domain mutations: Alterations in residues directly involved in hydroxylation activity can reveal the molecular basis of enzyme function

• Substrate binding site mutations: Modifications affecting collagen peptide recognition can elucidate substrate specificity determinants

• Protein-protein interaction interface mutations: Changes at interfaces with P4HB or other partners can reveal assembly requirements

When conducting mutational studies, employ a systematic approach:

• Target evolutionarily conserved residues identified through sequence alignment

• Focus on domains with known functional importance

• Include both alanine scanning and directed mutations based on structural predictions

• Assess effects on multiple parameters (activity, stability, localization)

Mutation effects should be quantified through enzymatic activity assays, thermal stability measurements, and interaction studies. Correlating mutational data with existing structural information from related proteins can generate refined models of P4HA3 function .

How can P4HA3 gene expression patterns be accurately quantified in mouse tissues?

For precise quantification of mouse P4HA3 expression across tissues:

• RT-qPCR analysis:

  • Design primers specific to mouse P4HA3 avoiding cross-reactivity with other P4HA isoforms

  • Validate primers using standard curves and melt curve analysis

  • Normalize to multiple reference genes (typically 3-4) selected for stability in target tissues

• RNA-Seq approach:

  • Provides comprehensive transcriptome analysis including splice variants

  • Requires appropriate bioinformatic pipelines for accurate quantification

  • Enables discovery of novel regulatory relationships

• In situ hybridization:

  • Allows cellular and subcellular localization of P4HA3 transcripts

  • RNAscope technology provides single-molecule sensitivity

• Single-cell RNA-Seq:

  • Reveals cell type-specific expression patterns within heterogeneous tissues

  • Particularly valuable for complex tissues like tumors

What are the appropriate cell-based assays to study mouse P4HA3 functions?

To effectively study mouse P4HA3 functions in cellular contexts:

• Overexpression systems:

  • Transfect cells with mouse P4HA3 expression vectors

  • Validate expression by western blot and RT-qPCR

  • Assess effects on collagen production, secretion, and modification

• Knockdown/knockout approaches:

  • Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate P4HA3

  • Validate knockdown efficiency at protein and mRNA levels

  • Examine phenotypic consequences on cell migration, invasion, and ECM production

• Functional assays:

  • Collagen secretion assays using radiolabeled proline

  • Cell migration and invasion assays in 3D matrices

  • Extracellular matrix stiffness measurements

  • Hypoxia response element (HRE) reporter assays

• Co-culture systems:

  • Combine P4HA3-modified cells with immune cells to study interactions

  • Evaluate effects on endothelial cells to assess angiogenic potential

Each assay should include appropriate controls, including rescue experiments to confirm specificity of observed effects .

How can recombinant mouse P4HA3 be used in the development of targeted therapeutics?

Recombinant mouse P4HA3 has several applications in therapeutic development:

• High-throughput screening:

  • Enzymatic assays using recombinant P4HA3 to identify inhibitors

  • Structure-based virtual screening leveraging protein structural information

  • Fragment-based approaches to develop highly specific inhibitors

• Antibody development:

  • Immunization with recombinant P4HA3 to generate monoclonal antibodies

  • Phage display screening against purified protein

  • Validation of antibody specificity and efficacy in mouse models

• Therapeutic validation:

  • Testing candidate compounds in P4HA3-dependent cellular assays

  • Evaluating effects on collagen modification and tumor progression

  • Comparing effects between species to predict translational potential

• Target engagement studies:

  • Cellular thermal shift assays (CETSA) to confirm binding in cellular context

  • Competition binding assays with labeled probe compounds

  • Microscopy-based co-localization studies

When developing P4HA3-targeted therapeutics, consider isoform specificity to avoid off-target effects on the related P4HA1 and P4HA2 proteins, which may have distinct physiological roles .

How does mouse P4HA3 contribute to cancer progression mechanisms?

Mouse P4HA3 influences cancer progression through multiple mechanisms:

• Extracellular matrix remodeling:

  • Promotes collagen cross-linking and fibril formation

  • Increases matrix stiffness, enhancing cancer cell migration and invasion

  • Creates tracks for cancer cell movement through tissues

• Hypoxia response:

  • Functions as part of cellular adaptation to hypoxic conditions

  • Correlates with hypoxia-inducible factor (HIF) pathway activation

  • Contributes to cancer cell survival under hypoxic tumor conditions

• Metastatic capacity:

  • Facilitates invasion through basement membrane

  • Enhances cancer cell survival in circulation

  • Contributes to pre-metastatic niche formation

• Immune modulation:

  • Alters immune cell infiltration patterns

  • Influences T-cell activation and function

  • Creates immunosuppressive microenvironment

What is the relationship between P4HA3 expression and immune cell infiltration in mouse tumor models?

Analysis of mouse tumor models reveals complex relationships between P4HA3 expression and immune infiltration:

• Correlation with immune cell populations:

  • Significant positive correlation with tumor-associated macrophages

  • Variable relationships with CD8+ T-cell infiltration depending on tumor type

  • Association with neutrophil recruitment in certain contexts

• Impact on immune checkpoint expression:

  • Positive correlation with PD-L1 expression in multiple tumor types

  • Association with other immune checkpoint molecules

• Influence on cytokine/chemokine profiles:

  • Altered expression of chemokines directing immune cell trafficking

  • Modulation of inflammatory cytokine production

  • Changes in immune signaling network

Research using single-cell RNA sequencing in mouse tumor models has further revealed cell type-specific effects of P4HA3 on the tumor immune microenvironment, suggesting potential implications for immunotherapy response .

How can gene correlation analysis be used to identify P4HA3-related pathways in cancer?

Gene correlation analysis provides valuable insights into P4HA3-associated pathways:

• Co-expression network analysis:

  • Identify genes with similar expression patterns to P4HA3

  • Construct functional modules through weighted gene co-expression network analysis (WGCNA)

  • Determine hub genes within P4HA3-associated networks

• Pathway enrichment analysis:

  • Apply Gene Set Enrichment Analysis (GSEA) to P4HA3-correlated genes

  • Identify enriched biological processes, molecular functions, and cellular components

  • Discover signaling pathways significantly associated with P4HA3 expression

• Regulatory relationship inference:

  • Predict transcription factors controlling P4HA3 expression

  • Identify microRNAs potentially regulating P4HA3 mRNA

  • Discover epigenetic mechanisms governing P4HA3 expression

These analyses have revealed that P4HA3 expression strongly correlates with extracellular matrix organization, hypoxia response, angiogenesis, and epithelial-to-mesenchymal transition pathways in multiple cancer types .

What are common challenges in producing active recombinant mouse P4HA3 and how can they be addressed?

Researchers frequently encounter several challenges when producing recombinant mouse P4HA3:

• Protein solubility issues:

  • Challenge: Formation of inclusion bodies in bacterial expression systems

  • Solution: Optimize induction conditions (lower temperature, reduced IPTG concentration)

  • Alternative: Use solubility tags (SUMO, MBP, TRX) or refolding protocols

• Incorrect folding:

  • Challenge: Improper disulfide bond formation affecting activity

  • Solution: Express in eukaryotic systems with appropriate oxidative environment

  • Alternative: Co-express with chaperones or protein disulfide isomerases

• Co-factor incorporation:

  • Challenge: Incomplete incorporation of iron into the catalytic site

  • Solution: Supplement growth media with iron and optimize purification buffers

  • Alternative: Reconstitute with iron post-purification

• Tetramer formation:

  • Challenge: Inefficient assembly with P4HB subunits

  • Solution: Co-express alpha and beta subunits in appropriate stoichiometry

  • Alternative: In vitro reconstitution of tetrameric complex

For highest activity, mammalian expression systems typically yield the most functionally active protein, though at lower yields than bacterial systems .

How can specificity of P4HA3 antibodies be validated for mouse studies?

Thorough validation of antibodies for mouse P4HA3 is essential:

• Western blot validation:

  • Test against recombinant mouse P4HA3

  • Compare with tissues from P4HA3 knockout mice (negative control)

  • Evaluate cross-reactivity with other P4H family members

  • Perform peptide competition assays

• Immunohistochemistry validation:

  • Compare staining patterns in wild-type vs. knockout tissues

  • Validate subcellular localization (primarily endoplasmic reticulum)

  • Perform appropriate antigen retrieval optimization

  • Include isotype controls

• Cross-species reactivity assessment:

  • Determine specificity for mouse vs. human/rat P4HA3

  • Identify epitopes that are species-specific or conserved

• Application-specific validation:

  • Validate separately for each application (WB, IHC, IP, etc.)

  • Determine optimal antibody concentration for each method

  • Document lot-to-lot consistency

When publishing, include comprehensive details of antibody validation to ensure reproducibility .

What experimental controls are essential when studying P4HA3 function in mouse models?

Rigorous experimental controls are critical when investigating P4HA3:

• Genetic model controls:

  • Littermate controls for genetic knockout studies

  • Empty vector controls for overexpression studies

  • Scrambled/non-targeting siRNA for knockdown experiments

  • Rescue experiments to confirm phenotype specificity

• Specificity controls:

  • Include other P4H family members (P4HA1, P4HA2) to distinguish isoform-specific effects

  • Use catalytically inactive mutants to separate enzymatic from scaffolding functions

  • Employ domain-specific deletions to map functional regions

• Physiological relevance controls:

  • Compare effects under normoxic and hypoxic conditions

  • Evaluate outcomes in different cell types and tissue contexts

  • Assess age-dependent effects in animal models

• Technical controls:

  • Include positive controls for activity assays

  • Measure multiple outputs to comprehensively assess function

  • Use multiple independent methods to validate key findings

What are promising approaches for developing mouse P4HA3-specific inhibitors?

Developing mouse P4HA3-specific inhibitors represents an important research direction:

• Structure-based design approaches:

  • Utilize homology models based on related prolyl hydroxylase structures

  • Identify unique binding pockets distinguishing P4HA3 from other isoforms

  • Apply molecular docking and virtual screening methodologies

• High-throughput screening strategies:

  • Develop miniaturized enzymatic assays compatible with large compound libraries

  • Implement cellular screening systems using P4HA3-dependent readouts

  • Apply fragment-based approaches to identify building blocks for inhibitor design

• Allosteric inhibitor development:

  • Target regulatory sites outside the catalytic domain

  • Disrupt protein-protein interactions essential for function

  • Identify compounds affecting oligomeric assembly

• Targeted degradation approaches:

  • Design P4HA3-specific PROTACs (proteolysis targeting chimeras)

  • Develop antibody-drug conjugates targeting P4HA3-expressing cells

  • Explore RNA-based approaches (siRNA, antisense oligonucleotides)

The most promising approaches will combine computational prediction with experimental validation to achieve both potency and specificity .

How might single-cell analysis advance understanding of P4HA3 function in mouse models?

Single-cell technologies offer transformative potential for P4HA3 research:

• Single-cell RNA sequencing applications:

  • Identify cell populations expressing P4HA3 at high resolution

  • Map co-expression networks in specific cell types

  • Track dynamic changes in P4HA3 expression during disease progression

• Spatial transcriptomics approaches:

  • Correlate P4HA3 expression with spatial location in tissues

  • Reveal relationships between P4HA3-expressing cells and microenvironmental features

  • Map cell-cell communication networks involving P4HA3-expressing cells

• Multi-omics integration:

  • Combine transcriptomic, proteomic, and epigenomic data at single-cell level

  • Identify regulatory mechanisms controlling cell-specific P4HA3 expression

  • Discover novel functions through correlation with cellular phenotypes

• Lineage tracing applications:

  • Track the fate of P4HA3-expressing cells during development and disease

  • Identify cellular origins of P4HA3-high populations in pathological conditions

  • Study clonal evolution of P4HA3-expressing cells

These approaches can reveal previously unrecognized heterogeneity in P4HA3 expression and function across different cellular contexts .

What potential exists for P4HA3 as a therapeutic target in mouse models of human disease?

P4HA3 holds substantial promise as a therapeutic target in multiple disease contexts:

• Cancer applications:

  • Target to reduce tumor stiffness and inhibit invasion

  • Combine with immunotherapy to enhance immune cell infiltration

  • Potential to reduce metastatic capacity

  • May sensitize tumors to chemotherapy by altering drug penetration

• Fibrotic disease models:

  • Potential to reduce excessive collagen deposition

  • May improve tissue function in fibrotic organs

  • Could complement existing anti-fibrotic approaches

• Wound healing applications:

  • Modulate collagen maturation to improve wound strength

  • Potential to reduce scarring through controlled collagen organization

  • May enhance healing in compromised wound environments

• Combination therapy approaches:

  • Pair with matrix metalloproteinase inhibitors for comprehensive ECM modulation

  • Combine with angiogenesis inhibitors to target tumor microenvironment

  • Use alongside immunomodulatory agents to enhance therapeutic efficacy