Recombinant Human Prokineticin-1 protein (PROK1) (Active)

What is the structural characterization of recombinant human PROK1?

Recombinant human PROK1 is a secreted protein characterized by a conserved N-terminal sequence (AVITGA) that is essential for its biological activity . The protein contains structural motifs that enable receptor binding and downstream signaling. When working with recombinant PROK1, it's important to verify its structural integrity through techniques such as:

• SDS-PAGE for molecular weight confirmation

• Western blot analysis using specific antibodies

• Mass spectrometry for detailed structural analysis

• Circular dichroism for secondary structure assessment

Research has demonstrated that the N-terminal hexapeptide sequence is critical for receptor recognition and activation, and modifications to this region can significantly alter protein function .

What are the primary signaling pathways activated by PROK1?

PROK1 primarily signals through its receptor PROKR1, activating multiple downstream pathways. Research has established that PROK1-PROKR1 interaction induces:

• Inositol phosphate mobilization

• Sequential phosphorylation of c-Src

• Activation of epidermal growth factor receptor (EGFR)

• ERK 1/2 phosphorylation

• Calcium-calcineurin signaling pathway activation in a G-protein (Gq/11) dependent manner

These signaling cascades culminate in the regulation of target genes, including cyclooxygenase-2 (COX-2), leukemia inhibitory factor, IL-6, IL-8, and IL-11, which are crucial for processes such as implantation and early pregnancy . In experimental settings, inhibitors targeting these pathways can be used to delineate the specific contributions of each signaling component to PROK1-mediated effects.

What are the optimal conditions for in vitro applications of recombinant PROK1?

For optimal results in cell-based assays, recombinant PROK1 should be used under the following conditions:

• Concentration: Most studies utilize 40 nM PROK1 for in vitro experiments, which has been shown to effectively induce downstream responses

• Medium: Serum-free medium is recommended for treatments to avoid interference from serum components

• Pre-treatment period: Cells should be serum-starved overnight before PROK1 administration

• Treatment duration: Effects on signaling can be observed within minutes, while gene expression changes typically require 6-8 hours of treatment

For in vivo studies in mouse models, a dose of 350 nM has been used for intrauterine injections, which is approximately ten times the dose that induces cytokine expression in human gestational tissues .

How can researchers verify PROK1 activity in experimental systems?

To confirm the biological activity of recombinant PROK1 in experimental systems, researchers should implement multiple validation strategies:

• Receptor binding assays: Verify interaction with PROKR1 using cells expressing the receptor

• Signaling pathway activation: Measure phosphorylation of downstream targets (ERK 1/2, c-Src) using Western blotting

• Calcium mobilization assays: Assess intracellular calcium flux using fluorescent indicators

• Target gene expression: Quantify induction of known PROK1-regulated genes (e.g., IL-11, COX-2) using RT-PCR or qPCR

• Functional assays: Evaluate biological responses such as prostaglandin production using ELISA methods

Researchers should include appropriate positive and negative controls, including PROKR1-expressing and non-expressing cell lines, to ensure result validity.

How can researchers effectively modulate PROK1 signaling in experimental models?

Several sophisticated approaches can be employed to modulate PROK1 signaling for mechanistic studies:

Genetic approaches:

• CRISPR/Cas9-mediated knockout of PROK1 or PROKR1

• RNA interference using siRNA or miRNA targeting PROK1, as demonstrated in studies of first trimester decidua

• Overexpression systems using viral vectors (lentivirus or adenovirus) for gain-of-function studies

Pharmacological approaches:

• Specific pathway inhibitors targeting calcium signaling, ERK, or calcineurin pathways

• Dominant-negative constructs of signaling components (c-Src, EGFR, Ras, and MEK) to disrupt specific pathway nodes

• RCAN1-4 (regulator of calcineurin 1 isoform 4) overexpression to negatively regulate calcineurin signaling, which has been shown to reduce PROK1-induced IL-11 expression

For tissue-specific manipulations, researchers have successfully used viral delivery systems with titers exceeding 1 × 10^10 IFU/ml, added at five adenovirus pfu/plated cell .

What methodologies are most effective for studying PROK1's role in pregnancy and implantation?

Given PROK1's significant role in pregnancy and implantation, several specialized methodologies have proven effective:

In vitro models:

• Endometrial epithelial cell lines stably expressing PROKR1 (e.g., Ishikawa PROKR1 cells)

• Primary cell cultures from first-trimester decidua

• Co-culture systems mimicking maternal-fetal interface

In vivo approaches:

• Mouse models of term and preterm parturition using intrauterine injection techniques

• Timed-pregnant models with surgical interventions for PROK1 administration

• Dual-immunofluorescence histochemistry for colocalization studies of PROK1 with markers such as CD56 (natural killer cells) or PROKR1 with COX-2 or CD31 (endothelial cells)

When using mouse models, researchers have successfully employed intrauterine injection between the two most anterior fetuses using a 33-gauge Hamilton syringe with 25 μl volume of recombinant PROK1 (350 nM) to study effects on pregnancy outcomes .

How can researchers differentiate between direct and indirect effects of PROK1 in complex tissues?

Differentiating direct from indirect PROK1 effects requires sophisticated experimental designs:

• Cell-type specific analyses:

  • Laser capture microdissection to isolate specific cell populations from heterogeneous tissues

  • Single-cell RNA sequencing to identify cell-specific responses

  • Immunohistochemistry with dual or triple labeling to identify responding cell types

• Temporal studies:

  • Time-course experiments to distinguish primary from secondary responses

  • Pulse-chase protocols to track signaling propagation

  • Conditional expression systems for temporal control of PROK1 expression

• Pathway dissection:

  • Selective inhibition of downstream mediators to block specific effector pathways

  • Receptor antagonism studies to confirm PROKR1-dependent effects

  • Transcriptional profiling with and without protein synthesis inhibitors to distinguish direct transcriptional targets from secondary gene expression changes

Research has demonstrated that PROK1 induces expression of IL-11 via the calcineurin signaling pathway in a G-protein (Gq/11), calcium, and ERK-dependent manner, highlighting the complexity of these pathways .

What are the current challenges in measuring PROK1 levels in biological specimens?

Researchers face several technical challenges when quantifying PROK1 in biological samples:

Analytical challenges:

• Low abundance in certain tissues requiring sensitive detection methods

• Potential cross-reactivity with related proteins (e.g., PROK2)

• Post-translational modifications affecting antibody recognition

• Matrix effects in complex biological samples (serum, tissue extracts)

Methodological solutions:

• Use of ELISA methods optimized for specific sample types (e.g., serum samples for maternal PROK1 levels)

• Validation with multiple antibodies targeting different epitopes

• Sample pre-treatment to remove interfering substances

• Inclusion of appropriate standard curves with recombinant protein

• Western blot confirmation of ELISA results where possible

A standardized approach is particularly important when investigating correlations between PROK1 levels and clinical outcomes, such as in studies of maternal serum PROK1 in relation to pregnancy complications in women with polycystic ovary syndrome .

How can gene expression changes induced by PROK1 be comprehensively analyzed?

To thoroughly analyze PROK1-induced gene expression changes, researchers should employ multiple complementary approaches:

Global profiling techniques:

• RNA sequencing or microarray analysis to identify all differentially regulated genes

• Proteomics to assess changes at the protein level

• Pathway analysis tools to identify enriched functional categories

Validation strategies:

• RT-PCR or qPCR for candidate gene verification

• Western blotting to confirm protein level changes

• Functional assays to assess biological relevance

Gene microarray analysis on RNA from Ishikawa PROKR1 cells treated with 40 nM PROK1 for 8 hours revealed 49 differentially regulated genes, many of which are involved in implantation and early pregnancy . For targeted analysis of specific PROK1-regulated genes in experimental tissues, researchers typically harvest cells or tissues for RNA extraction after PROK1 treatment, with or without pathway inhibitors, followed by PCR analysis .

What controls should be included in PROK1 signaling studies?

Robust PROK1 research requires comprehensive controls:

Essential experimental controls:

• Vehicle controls for recombinant protein treatments (e.g., saline or buffer only)

• Receptor-negative cells to confirm PROKR1-dependent effects

• Pathway inhibitor controls with concentration-response assessment

• Time-matched controls for temporal studies

• Negative control antibodies (rabbit IgG) for immunohistochemistry

Tissue-specific considerations:

• For pregnancy-related studies, appropriate gestational age-matched controls are essential

• When investigating cellular localization, include both cell-type specific markers (CD31, CD56) and negative controls for co-localization studies

• For in vivo studies, sham-operated controls and non-pregnant controls should be included as appropriate

In mouse studies of PROK1's effects on parturition, researchers have used saline injection controls and included measurements from natural delivery (without surgical intervention) for proper comparative analysis .

How should researchers approach contradictory findings in PROK1 studies?

When confronted with seemingly contradictory findings in PROK1 research, a systematic approach is recommended:

• Methodological reconciliation:

  • Compare experimental conditions (concentrations, timing, cell types)

  • Assess differences in readouts and their sensitivity

  • Evaluate model systems (in vitro vs. in vivo, species differences)

• Biological context considerations:

  • PROK1 effects may be tissue and context-dependent

  • Receptor expression levels can dramatically alter responses

  • Feedback mechanisms may operate differently across systems

• Reproducibility assessment:

  • Replicate key experiments with variations in conditions

  • Use multiple methodological approaches to measure the same outcome

  • Calculate statistical power to ensure adequate sample sizes

For example, in studies of PROK1's effect on preterm labor in mice, researchers found that while PROK1 induced pro-inflammatory responses and shortened the time to delivery by 28%, this effect was less than half as powerful as LPS and did not reach statistical significance. A post hoc power calculation revealed that 21 mice per group would be needed to detect this difference with 90% power .

What statistical approaches are most appropriate for analyzing PROK1 data?

Appropriate statistical analysis of PROK1 data depends on experimental design:

For expression studies:

• For normally distributed data: ANOVA with post-hoc tests (e.g., Fisher's protected least significant difference test)

• For non-normally distributed data: Non-parametric tests (Wilcoxon, Mann-Whitney)

• For time-course studies: Repeated measures ANOVA or mixed-effects models

For clinical correlations:

• Multiple regression to account for confounding variables

• Logistic regression for binary outcomes

• Receiver operating characteristic (ROC) curve analysis to assess predictive value

Power calculations:

• Essential for determining adequate sample sizes

• Particularly important in animal studies and clinical research

• Should be reported transparently in publications

In mouse studies of PROK1, post hoc power calculations indicated that 21 mice per group would be needed to detect a 28% reduction in time to delivery with 90% power at a 5% significance level . This highlights the importance of power calculations in interpreting negative results.

What emerging technologies could advance PROK1 research?

Several cutting-edge technologies hold promise for advancing PROK1 research:

Emerging methodologies:

• Single-cell multi-omics for comprehensive cellular response profiling

• CRISPR screening to identify novel regulators and targets of PROK1 signaling

• Optogenetic control of PROK1 signaling for precise temporal manipulation

• Organ-on-chip models of the maternal-fetal interface for studying PROK1 in implantation

• Spatial transcriptomics to map PROK1 effects across tissue microenvironments

Analytical innovations:

• Machine learning approaches to integrate multi-dimensional PROK1 data

• Systems biology modeling of PROK1 signaling networks

• High-content imaging with multiplexed antibody staining for pathway analysis

These technologies could help address current knowledge gaps, such as cell-specific responses to PROK1 in heterogeneous tissues and the integration of PROK1 signaling with other regulatory pathways.

How might therapeutic applications of PROK1 research develop in the future?

While avoiding commercial aspects, several research directions could inform future therapeutic applications:

Potential research targets:

• Development of highly specific PROKR1 agonists and antagonists for experimental use

• Investigation of PROK1's role in modulating inflammatory responses in reproductive tissues

• Exploration of the relationship between PROK1 signaling and pregnancy complications

Translational considerations:

• Further elucidation of PROK1's role in implantation could inform research on implantation failure

• Understanding PROK1's pro-inflammatory effects might provide insights into preterm labor mechanisms

• Research on serum PROK1 levels might contribute to biomarker development for pregnancy complications

Studies have shown that maternal serum PROK1 in the second trimester did not predict pregnancy complications in women with PCOS, but interestingly, PROK1 levels were lower in hyperandrogenic women and in those using metformin . These findings suggest complex regulatory mechanisms that warrant further investigation.