Recombinant Macaca fascicularis P2Y purinoceptor 12 (P2RY12)

What expression systems are available for producing recombinant Macaca fascicularis P2RY12?

Recombinant Macaca fascicularis P2RY12 can be produced using multiple expression systems, each with distinct advantages depending on research requirements:

For most basic research applications, the E. coli-expressed full-length protein (CSB-CF839200MOV) is suitable, while studies requiring authentic post-translational modifications may benefit from mammalian cell expression systems .

How should recombinant Macaca fascicularis P2RY12 be stored and reconstituted for optimal activity?

For optimal preservation of recombinant Macaca fascicularis P2RY12 activity, follow these evidence-based storage and reconstitution protocols:

• Long-term storage: Store lyophilized powder at -20°C/-80°C upon receipt.

• Aliquoting: Divide reconstituted protein into working aliquots to avoid repeated freeze-thaw cycles.

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (recommended: 50%)

• Working storage: Store working aliquots at 4°C for up to one week

• Buffer composition: Tris/PBS-based buffer, 6% Trehalose, pH 8.0

Important note: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided .

How does neuronal expression of P2RY12 in Macaca fascicularis differ under metabolic stress conditions?

• Obese and diabetic cynomolgus monkeys (Macaca fascicularis) demonstrate altered expression patterns of NF-κB-associated transcripts in PVHOXT neurons

• This suggests NF-κB pathway involvement in upregulating P2Y12, its direct downstream target

• The "ectopic" expression of P2Y12 on OXT neurons has been detected in metabolically stressed states

• This neuronal expression pattern coincides with elevation of ATP inflares, constituting a novel purinergic pathway in the metabolically stressed hypothalamus

• In experimental models, lifting caloric restriction in aged monkeys while administering P2Y12 blockers prevented obesity development

These findings suggest P2RY12 may play a previously unrecognized role in metabolic regulation in primates, with potentially significant implications for understanding and treating metabolic disorders in humans .

What methodological considerations are important when designing functional assays for P2RY12 signaling using the recombinant protein?

When designing functional assays for P2RY12 signaling using recombinant Macaca fascicularis protein, researchers should consider these critical methodological factors:

• Receptor reconstitution: For membrane proteins like P2RY12, lipid environment significantly impacts function. Consider:

  • Detergent selection during purification (mild non-ionic detergents preserve activity)

  • Reconstitution into proteoliposomes or nanodiscs for functional studies

  • Membrane composition matching target tissue (brain vs. platelets)

• Signaling cascade measurement:

  • Gi-protein coupling assays measuring inhibition of adenylyl cyclase

  • cAMP detection assays (decreased cAMP levels indicate receptor activation)

  • Cell-based reporter systems expressing recombinant P2RY12 (e.g., GT1-7 cells)

• Agonist selection and concentration:

  • ADP as primary physiological agonist (concentration range: 1 nM - 10 μM)

  • ATP as potential competitive antagonist at certain concentrations

  • Control for nucleotide degradation during assay incubation

• Controls and validation:

  • Positive control: Verify system using known P2RY12 agonists

  • Negative control: Include P2RY12 antagonists (ticagrelor, cangrelor)

  • Species comparison: Human P2RY12 vs. Macaca fascicularis P2RY12

In cell-based systems, P2RY12 overexpression has been shown to reduce intracellular cAMP levels and diminish Fos expression, thereby antagonizing MC4R/Gs signaling in melanocortin system cells . This can serve as a functional readout for proper receptor activity.

What are the key differences between human and Macaca fascicularis P2RY12 that may impact translational research?

Understanding the similarities and differences between human and Macaca fascicularis P2RY12 is crucial for translational research:

These differences should be considered when using Macaca fascicularis P2RY12 as a model for human P2RY12 in drug development or disease modeling .

What purification strategies are most effective for isolating high-quality recombinant Macaca fascicularis P2RY12?

Purifying high-quality recombinant Macaca fascicularis P2RY12 requires specific strategies to overcome challenges associated with membrane protein isolation:

• Tag selection and position:

  • N-terminal His-tag is commonly used with minimal impact on function

  • Tag position matters: C-terminal tags may interfere with G-protein coupling

  • For specific applications, consider alternative tags (FLAG, Strep-II)

• Optimized extraction protocol:

  • Detergent screening critical (start with n-dodecyl-β-D-maltoside, CHAPS, or LMNG)

  • Extraction temperature: 4°C recommended to prevent protein degradation

  • Extraction time: 2-4 hours optimal for most preparations

• Multi-step purification strategy:
  a. Immobilized metal affinity chromatography (IMAC)

  • Use Ni-NTA or TALON resin for His-tagged proteins

  • Include low imidazole (10-20 mM) in wash buffers to reduce non-specific binding
    b. Size exclusion chromatography (SEC)

  • Critical for removing aggregates and ensuring homogeneity

  • Buffer composition: typically Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• Quality control assessments:

  • SDS-PAGE: >90% purity criterion for most applications

  • Western blot: Confirm identity using anti-P2RY12 or anti-His antibodies

  • SEC-MALS: Verify monodispersity and appropriate molecular weight

Using this approach, researchers can consistently achieve >90% purity as determined by SDS-PAGE, making the preparation suitable for most research applications .

How can researchers effectively validate the functionality of recombinant Macaca fascicularis P2RY12 in experimental settings?

Comprehensive validation of recombinant Macaca fascicularis P2RY12 functionality requires multiple complementary approaches:

• Ligand binding assays:

  • Radioligand binding using [³H]ADP or [³H]2MeS-ADP

  • Surface plasmon resonance (SPR) with immobilized receptor

  • Microscale thermophoresis for label-free affinity determination

  • Expected binding parameters: Kd for ADP = 10-100 nM range

• G-protein coupling assays:

  • GTPγS binding assays to measure G-protein activation

  • BRET/FRET-based assays for real-time monitoring of G-protein dissociation

  • Co-immunoprecipitation of receptor with Gαi subunits

• Downstream signaling validation:

  • Measurement of inhibition of adenylyl cyclase activity

  • Quantification of reduced cAMP levels (using ELISA or FRET-based sensors)

  • Verification of reduced Fos expression in responsive cell lines

• Cell-based functional assays:

  • Transfection into GT1-7 cells (as demonstrated in research)

  • Measurement of α-MSH-induced cAMP responses (should be diminished)

  • Assessment of transcriptional responses to receptor activation

Researchers have successfully validated P2RY12 functionality by demonstrating that overexpression in hypothalamic GT1-7 cells reduces intracellular cAMP levels and diminishes Fos expression, leading to an inability of α-MSH to elicit transcriptional response in these cells .

What are the best approaches for studying P2RY12 antagonism in Macaca fascicularis models of metabolic disorders?

Recent research has established effective approaches for studying P2RY12 antagonism in Macaca fascicularis models of metabolic disorders:

• Animal model selection and preparation:

  • Focus on aged Macaca fascicularis (15% naturally develop obesity when provided ad libitum food)

  • Baseline characterization: Monitor food intake, body weight, and metabolic parameters

  • Consider calorie restriction lifting paradigm: Increase chow diet availability by 80% while co-administering P2RY12 antagonists

• P2RY12 antagonist selection:

  • Irreversible inhibitor prodrugs: ticlopidine (90 mg/kg/day, 27x human dose)

  • Direct-acting inhibitors: ticagrelor, cangrelor

  • Administration routes: oral, intranasal (demonstrated BBB penetration)

  • Verify antagonist bioavailability: Monitor presence in hypothalamus (e.g., via mass spectrometry)

• Outcome measurements:

  • Primary outcomes: Food intake, body weight, glucose tolerance, insulin sensitivity

  • Secondary measures: Fasting glucose levels, insulin tolerance test performance

  • Molecular readouts: Expression of P2RY12 in hypothalamic neurons, cAMP levels

• Experimental design considerations:

  • Duration: Extended monitoring (even after withdrawal of treatment)

  • Controls: Both vehicle-treated and untreated groups

  • Sample collection: Consider post-mortem tissue analysis for neuronal P2RY12 expression

Research has demonstrated that P2Y12 blockers can prevent obesity development in Macaca fascicularis even after withdrawal of administration, with effects on normalizing food intake and improving metabolic parameters .

How does "ectopic" P2RY12 expression in neurons impact neuronal function and metabolic regulation?

The recently discovered phenomenon of "ectopic" P2RY12 expression in neurons under metabolic stress opens new research avenues:

• Mechanistic basis of ectopic expression:

  • HFD (high-fat diet) induces expression of P2RY12 on 54.67 ± 3.62% of PVHOXT neurons vs. none in chow-fed animals

  • FACS-isolated PVHOXT neurons from HFD-fed mice show drastic upregulation of P2ry12 transcript

  • NF-κB pathway appears to drive this upregulation as a direct downstream target

  • Similar patterns observed in human patients with diabetes mellitus (3 out of 4 investigated patients)

• Functional consequences on neuronal activity:

  • P2RY12 activation leads to Gi-dependent inactivation of adenylyl cyclase/cAMP/c-Fos axis

  • This antagonizes the MC4R/Gs signaling in second-order melanocortin system cells

  • P2RY12 overexpression in hypothalamic cells reduces intracellular cAMP and diminishes α-MSH response

  • Net effect appears to be altered energy homeostasis promoting obesity

• Therapeutic implications:

  • P2RY12 antagonists (ticlopidine, ticagrelor, cangrelor) can reverse obesity in animal models

  • Effects persist even after withdrawal of treatment

  • Both food intake and glucose/insulin parameters improve with treatment

This discovery represents a novel purinergic pathway in the metabolically stressed hypothalamus with significant implications for understanding and treating metabolic disorders .

What research approaches can effectively explore cross-species conservation of P2RY12 function between Macaca fascicularis and humans?

To effectively investigate cross-species conservation of P2RY12 function, researchers should consider these complementary approaches:

• Comparative genomics and structural biology:

  • Sequence alignment of human and Macaca fascicularis P2RY12 coding and regulatory regions

  • Homology modeling based on crystal structures

  • Molecular dynamics simulations to identify functionally conserved domains

  • Focus on ligand binding pocket and G-protein interaction interfaces

• Pharmacological profiling:

  • Comparative binding studies with identical ligand panels

  • Dose-response curves for agonists and antagonists across species

  • Allosteric modulator screening for species-specific effects

  • Drug metabolism and pharmacokinetic comparisons

• Signaling pathway conservation:

  • Side-by-side G-protein coupling efficiency measurements

  • Quantitative cAMP inhibition comparisons

  • Phosphoproteomic analysis of downstream effectors

  • Cross-species cell line models expressing each receptor variant

• Pathophysiological models:

  • Parallel disease models (e.g., metabolic disorders, thrombosis)

  • Response to identical interventions across species

  • Cross-validation of "ectopic" expression patterns in metabolic stress

  • Translational biomarkers applicable across species

Implementation of these approaches would provide comprehensive understanding of how findings in Macaca fascicularis models translate to human applications, particularly for metabolic disorders where P2RY12 plays newly discovered roles .

What technological advances might improve the production and structural characterization of recombinant Macaca fascicularis P2RY12?

Emerging technologies offer promising avenues to enhance production and structural characterization of recombinant Macaca fascicularis P2RY12:

• Advanced expression systems:

  • Cell-free expression systems for rapid production and direct incorporation into nanodiscs

  • Engineered mammalian cell lines with enhanced GPCR folding machinery

  • Directed evolution approaches to identify stabilizing mutations

  • Combinatorial fusion partner screening for improved expression

• Membrane mimetic technologies:

  • Saposin-based lipoprotein nanoparticles (Salipro)

  • Styrene maleic acid lipid particles (SMALPs) for native-like environments

  • Custom-designed nanodisc systems with optimized lipid compositions

  • Amphipol stabilization for enhanced thermostability

• Structural biology innovations:

  • Cryo-EM advances for membrane protein structures at near-atomic resolution

  • Integrative structural biology combining multiple techniques (NMR, X-ray, SAXS)

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural insights

  • Computational methods for predicting species-specific structural differences

• Functional validation technologies:

  • Label-free binding and functional assays with higher throughput

  • Single-molecule FRET for conformational dynamics studies

  • Electrical impedance measurements for receptor activation in real-time

  • Biosensor development for in vivo monitoring of P2RY12 activity

These technological advances would overcome current limitations in studying this challenging membrane protein, enabling more detailed understanding of its structure-function relationships and species-specific characteristics .

How should researchers interpret contradictory findings between in vitro and in vivo studies of Macaca fascicularis P2RY12?

When confronted with contradictory findings between in vitro and in vivo studies of Macaca fascicularis P2RY12, consider this systematic approach:

• System complexity differences:

  • In vitro systems lack the complex regulatory environment of living organisms

  • P2RY12 functions within purinergic signaling networks with multiple feedback mechanisms

  • Expression level differences between recombinant systems and native tissues

  • Recommendation: Map the complete signaling network in both systems to identify missing components

• Methodological considerations:

  • Different detection methods may have varying sensitivity and specificity

  • Recombinant protein modifications (tags, expression system) may alter function

  • Pharmacological tool compounds may have different off-target effects in vivo

  • Recommendation: Validate key findings using multiple methodological approaches

• Physiological context:

  • Nucleotide concentration differences between in vitro assays and tissue microenvironments

  • Cell type-specific signaling partners not present in reconstituted systems

  • "Ectopic" expression patterns in stress conditions alter normal function

  • Recommendation: Match experimental conditions to physiological parameters when possible

• Reconciliation strategies:

  • Design intermediate complexity models (e.g., tissue slices, organoids)

  • Use genetic approaches (CRISPR) to confirm pharmacological findings

  • Apply systems biology modeling to predict how in vitro mechanisms scale to in vivo

  • Consider species differences between model systems

Recent research demonstrated that while P2Y12 inhibitors like ticlopidine, clopidogrel, and prasugrel showed similar in vitro potency, only ticlopidine effectively reversed diet-induced obesity in vivo, highlighting the importance of pharmacokinetic differences and BBB penetration in translating in vitro findings .

What are the key considerations when comparing results from different expression systems for Macaca fascicularis P2RY12?

When comparing results from different expression systems for recombinant Macaca fascicularis P2RY12, researchers should carefully consider:

• Post-translational modification differences:

  Expression System	Glycosylation	Phosphorylation	Palmitoylation	Disulfide Bonds
  E. coli	Absent	Minimal	Absent	Often incorrect
  Yeast	Different pattern	Present	Partial	Usually correct
  Baculovirus	Present but simple	Present	Present	Correct
  Mammalian	Native-like	Native-like	Native-like	Correct

• Protein folding and quality:

  • Prokaryotic systems may produce inclusion bodies requiring refolding

  • Eukaryotic systems generally produce properly folded protein but at lower yields

  • Quality control metrics differ between systems (e.g., specific activity vs. total yield)

  • Recommendation: Include multiple quality control assays specific to each system

• Functional implications:

  • Ligand binding affinities may vary significantly between expression systems

  • G-protein coupling efficiency often depends on proper post-translational modifications

  • Detergent solubilization can differentially affect proteins from different sources

  • Thermostability profiles vary between expression systems

• Experimental design considerations:

  • Use standardized assay conditions when comparing across systems

  • Include reference standards with known activity

  • Consider developing correction factors for system-specific biases

  • Validate key findings using native tissue sources when possible

For optimal experimental outcomes, researchers should select expression systems based on specific experimental requirements rather than assuming equivalence across platforms .

How can researchers effectively troubleshoot experiments when recombinant Macaca fascicularis P2RY12 shows unexpected properties or poor activity?

When recombinant Macaca fascicularis P2RY12 exhibits unexpected properties or poor activity, follow this systematic troubleshooting approach:

• Protein quality assessment:

  • Verify purity via SDS-PAGE (should be >90%)

  • Check for degradation using Western blot with N and C-terminal antibodies

  • Assess aggregation state via size exclusion chromatography

  • Verify identity by mass spectrometry or N-terminal sequencing

  • Action: If quality issues identified, optimize purification protocol

• Storage and handling factors:

  • Avoid repeated freeze-thaw cycles (prepare single-use aliquots)

  • Ensure proper reconstitution (deionized sterile water to 0.1-1.0 mg/mL)

  • Add glycerol (5-50%, optimally 50%) for stability

  • Store working aliquots at 4°C for maximum one week

  • Action: Implement stricter storage protocol with stability monitoring

• Assay-specific considerations:

  • Nucleotide quality: Use fresh, high-purity ADP preparations

  • Buffer composition: Ensure physiological pH (7.4) and ionic strength

  • Divalent cations: Include proper Mg²⁺ concentration (1-2 mM)

  • Detergent interference: Minimize detergent concentration or switch to nanodiscs

  • Action: Systematically test assay components using positive controls

• Expression system-specific issues:

  • E. coli: Check for proper disulfide bond formation and refolding

  • Yeast/insect/mammalian: Verify glycosylation status

  • All systems: Confirm tag accessibility for purification

  • Action: Consider alternative expression system if issues persist

Experience has shown that recombinant P2RY12 activity is particularly sensitive to storage conditions, with significant activity loss occurring after multiple freeze-thaw cycles. Additionally, the choice of detergent for membrane protein stabilization critically impacts receptor functionality .