Recombinant Human Popeye domain-containing protein 3 (POPDC3)

What is the molecular structure of human POPDC3 protein?

Human POPDC3 is a transmembrane protein consisting of 291 amino acids with a unique cyclic adenosine monophosphate (cAMP) binding domain known as the Popeye domain. The protein contains transmembrane regions, with the amino acid sequence including characteristic hydrophobic segments that form membrane-spanning helices. The full sequence is: MERNSSLWKNLIDEHPVCTTWKQEAEGAIYHLASILFVVGFMGGSGFFGLLYVFSLLGLGFLCSAVWAWVDVCAADIFSWNFVLFVICFMQFVHIAYQVRSITFAREFQVLYSSLFQPLGISLPVFRTIALSSEVVTLEKEHCYAMQGKTSIDKLSLLVSGRIRVTVDGEFLHYIFPLQFLDSPEWDSLRPTEEGIFQVTLTAETDCRYVSWRRKKLYLLFAQHRYISRLFSVLIGSDIA DKLYALNDRVYIGKRYHYDIRLPNFYQMSTPEIRRSPLTQHFQNSRRYCDK .

What are the key functional domains of POPDC3?

POPDC3 contains several critical functional domains:

• The Popeye domain - a characteristic cAMP binding region that is unique to the POPDC family

• Transmembrane domains - allow proper membrane integration

• Carboxy-terminal domain - involved in protein-protein interactions

Mutations affecting highly conserved residues in the Popeye domain (such as p.Leu155His and p.Leu217Phe) and carboxy-terminal domain (p.Arg261Gln) have been associated with limb girdle muscular dystrophy, indicating their functional importance in skeletal muscle physiology .

How is POPDC3 expressed across different human tissues?

POPDC3 is widely expressed in mammalian tissues, with the highest levels of expression found in skeletal muscle. It is also present in cardiac tissue, though at lower levels. The differential expression pattern suggests tissue-specific functions, particularly in muscle tissues. This expression pattern makes it particularly relevant for studying muscle-related disorders and potentially as a biomarker in certain cancers where aberrant expression has been observed .

What expression systems are optimal for producing recombinant POPDC3?

There are two primary expression systems commonly used for recombinant POPDC3 production:

• Mammalian expression systems (HEK-293 cells) - Provides proper post-translational modifications and protein folding, resulting in proteins with >90% purity as determined by Bis-Tris PAGE. This system is particularly recommended for studies requiring functional interactions with mammalian proteins .

• Bacterial expression systems (E. coli) - More cost-effective but may lack some post-translational modifications. Still achieves >90% purity as determined by SDS-PAGE. This system is suitable for structural studies and applications where glycosylation is not critical .

The choice depends on your specific research needs - mammalian systems are preferred for functional studies, while bacterial systems may be sufficient for structural analyses.

What are the recommended storage conditions for recombinant POPDC3?

For optimal stability of recombinant POPDC3:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, add 5-50% glycerol (final concentration) with a recommended default of 50%

• Aliquot to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

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

• Centrifuge vials briefly before opening to bring contents to the bottom

Following these storage procedures minimizes protein degradation and maintains functional integrity for experimental use .

What purification methods yield the highest quality POPDC3 protein?

High-quality POPDC3 protein can be obtained through:

• One-step affinity chromatography - Using His-tag affinity purification for bacterial or mammalian expressed proteins

• Quality control methods - Including:

  • Bis-Tris PAGE or SDS-PAGE (>90% purity)

  • Anti-tag ELISA

  • Western Blot analysis

  • Analytical SEC (HPLC)

These methods ensure protein purity while maintaining structural integrity and functional activity. For experiments requiring extremely high purity, additional chromatography steps may be necessary .

How do POPDC3 gene variants contribute to limb-girdle muscular dystrophy?

POPDC3 gene variants cause a newly described form of limb-girdle muscular dystrophy (LGMD type 26) through several mechanisms:

• Specific missense mutations - Homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) affect highly conserved residues in the Popeye and carboxy-terminal domains

• Functional consequences - These mutations disrupt proper POPDC3 function in:

  • cAMP signaling

  • Improper modulation of the mechano-gated potassium channel TREK-1

  • Potential disruption of muscle membrane integrity

Patients with these variants present with:

• Proximal muscle weakness with adult-onset

• Lower limbs affected earlier than upper limbs

• Serum creatine kinase levels of 1,050 to 9,200 U/l

• Dystrophic changes on muscle biopsy

• Fat replacement of paraspinal and proximal leg muscles on MRI

This information is valuable for understanding how specific molecular defects in POPDC3 translate to clinical manifestations of muscular dystrophy .

What evidence supports POPDC3 as a biomarker for cancer radioresistance?

Multiple lines of evidence support POPDC3 as a potential biomarker for cancer radioresistance, particularly in head and neck squamous cell carcinoma (HNSCC):

These findings suggest that POPDC3 expression levels could serve as a biomarker to predict radiotherapy response in HNSCC patients, potentially guiding treatment decisions and identifying patients who might benefit from radiation dose intensification or alternative therapeutic approaches .

What is known about POPDC3's role in normal cardiac function?

While POPDC3 shows lower expression in cardiac tissue compared to skeletal muscle, studies in animal models suggest it may still contribute to cardiac function:

• POPDC3 is part of the POPDC family, which includes POPDC1 and POPDC2 that have established roles in cardiac function

• POPDC3 interacts with the mechano-gated potassium channel TREK-1, which regulates membrane potential in cardiac cells

• cAMP binding by POPDC3 may modulate cardiac cell signaling, potentially affecting contractility and electrical conduction

How can zebrafish models be utilized to study POPDC3 function?

Zebrafish models have proven valuable for investigating POPDC3 function:

• Knockdown approach:

  • Using splice-site blocking morpholinos targeting popdc3

  • Two different morpholinos can be used for validation and to minimize off-target effects

• Phenotypic assessment:

  • Knockdown results in larvae with tail curling

  • Histological analysis reveals dystrophic muscle features

  • Behavioral assays can quantify swimming deficits

• Advantages of zebrafish:

  • Rapid development

  • Optical transparency facilitating imaging

  • Ability to screen compounds for therapeutic potential

  • Conservation of POPDC3 function between zebrafish and humans

This model system provides an efficient way to study POPDC3's role in muscle development and function, and to screen potential therapeutic interventions before moving to more complex mammalian models .

What heterologous expression systems are suitable for studying POPDC3 interactions with ion channels?

Xenopus laevis oocytes represent an excellent heterologous expression system for studying POPDC3 interactions with ion channels, particularly TREK-1:

• Experimental approach:

  • Cloning wild-type and mutant POPDC3 sequences into expression vectors

  • Co-expression with TREK-1 channel

  • Two-electrode voltage clamp recordings to measure TREK-1 currents

  • Comparison of channel modulation between wild-type and mutant POPDC3

• Key findings:

  • All three POPDC3 mutants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) cause aberrant modulation of TREK-1

  • This suggests a mechanistic link between POPDC3 mutations and muscle dysfunction through altered ion channel regulation

• Advantages:

  • Large cell size facilitating microinjection and electrophysiological recording

  • Low background of endogenous channels

  • Ability to control expression levels

  • Reproducible functional measurements

This system has been instrumental in elucidating how POPDC3 mutations affect ion channel function, providing mechanistic insights into the pathophysiology of POPDC3-associated muscular dystrophy .

What bioinformatic tools are most effective for analyzing POPDC3 expression in cancer datasets?

Several bioinformatic tools have proven effective for analyzing POPDC3 expression in cancer datasets:

• The Cancer Genome Atlas (TCGA) - Provides comprehensive genomic, transcriptomic, and clinical data for various cancer types

• X-tile analysis - Determines optimal cut-off values for POPDC3 expression levels based on survival information

• Weighted Correlation Network Analysis (WGCNA) - Identifies hub genes and gene modules associated with radioresistance

• Database for Annotation, Visualization and Integrated Discovery (DAVID) - Performs functional pathway analysis, including Gene Ontology term enrichment and KEGG pathway analysis

• Protein-Protein Interaction (PPI) network analysis - Constructed using STRING database and visualized with Cytoscape to evaluate interactions

• Kaplan-Meier Plotter - Analyzes survival data based on POPDC3 expression levels

These tools collectively enable comprehensive analysis of POPDC3's role in cancer, particularly in identifying its potential as a prognostic biomarker and therapeutic target .

How can contradictory findings regarding POPDC3 function across different tissues be reconciled?

Reconciling contradictory findings regarding POPDC3 function across different tissues requires a multifaceted approach:

• Tissue-specific expression patterns:

  • Quantify expression levels across tissues using qPCR, RNA-seq, and protein quantification

  • Consider splice variants that may have tissue-specific distribution

• Interacting protein partners:

  • Perform co-immunoprecipitation and mass spectrometry in different tissues

  • Map tissue-specific interactomes that may explain functional differences

• Signaling pathway analysis:

  • Investigate tissue-specific signaling pathways that may interact with POPDC3

  • Consider the varying concentrations of cAMP across tissues

• Functional redundancy:

  • Assess expression of other POPDC family members (POPDC1, POPDC2) in tissues where POPDC3 mutations show minimal effect

  • Generate tissue-specific conditional knockout models to bypass developmental compensation

This methodological framework helps explain why POPDC3 mutations primarily affect skeletal muscle despite broader expression patterns and why cardiac phenotypes may be absent despite POPDC3 expression in cardiac tissue .

What are the key considerations for interpreting POPDC3 as a biomarker in cancer prognosis studies?

When interpreting POPDC3 as a biomarker in cancer prognosis studies, researchers should consider:

• Expression threshold determination:

  • Use X-tile analysis to establish clinically relevant cut-off values

  • Validate thresholds across independent cohorts

• Multivariate analysis:

  • Include established clinical variables (stage, grade, age, etc.) in Cox regression models

  • Calculate hazard ratios with 95% confidence intervals

  • Develop nomograms incorporating POPDC3 with other prognostic factors

• Cancer type specificity:

  • POPDC3's prognostic value may vary across cancer types

  • Head and neck squamous cell carcinoma shows strong association with radioresistance

• Biological context:

  • Consider the biological mechanisms by which POPDC3 might influence cancer progression

  • Evaluate potential contributions to treatment resistance, particularly radioresistance

• Technical considerations:

  • Account for tumor heterogeneity in sampling

  • Consider method of detection (RNA-seq, immunohistochemistry, etc.)

  • Standardize protocols for quantification

These methodological considerations are essential for robust interpretation of POPDC3 as a prognostic biomarker and for designing prospective validation studies .

What experimental designs best address the dual role of POPDC3 in muscle physiology and cancer?

To address POPDC3's dual role in muscle physiology and cancer, the following experimental designs are recommended:

• Comparative expression analysis:

  Tissue/Cell Type	Method	Parameters to Measure
  Normal muscle	RNA-seq & proteomics	Baseline expression, splice variants
  Dystrophic muscle	RNA-seq & proteomics	Expression changes, pathway alterations
  Normal epithelium	RNA-seq & proteomics	Baseline expression, splice variants
  Cancer cells	RNA-seq & proteomics	Expression changes, correlation with aggression

• CRISPR-based approaches:

  • Generate cell-type specific knockout models

  • Create isogenic cell lines with specific POPDC3 mutations

  • Perform rescue experiments with wild-type POPDC3

• Functional assays:

  • Measure membrane integrity in muscle cells vs. migration in cancer cells

  • Assess response to cAMP modulation in both contexts

  • Evaluate TREK-1 current in both cell types

• Animal models with tissue-specific manipulation:

  • Muscle-specific POPDC3 knockout or mutation

  • Cancer xenografts with POPDC3 modulation

  • Combination models to assess interactions

• Translational approaches:

  • Correlate POPDC3 variants in patients with both muscular and cancer phenotypes

  • Develop tissue-specific targeting strategies

This comprehensive experimental approach allows for systematic investigation of how the same protein contributes to distinct pathophysiological processes in different tissue contexts .

What are the most promising approaches for targeting POPDC3 therapeutically in muscular dystrophy?

Several promising therapeutic approaches for targeting POPDC3 in muscular dystrophy warrant further investigation:

• Gene therapy approaches:

  • AAV-mediated delivery of wild-type POPDC3 to affected muscles

  • CRISPR-Cas9 correction of specific POPDC3 mutations

  • Antisense oligonucleotides to modulate splicing if applicable

• Small molecule development:

  • High-throughput screening for compounds that stabilize mutant POPDC3

  • cAMP analogs that might compensate for altered binding in mutants

  • Allosteric modulators that restore POPDC3-TREK-1 interaction

• TREK-1 channel modulation:

  • Direct TREK-1 agonists to bypass defective POPDC3 regulation

  • Compounds that strengthen remaining POPDC3-TREK-1 interactions

• Signaling pathway interventions:

  • Modulation of downstream pathways affected by POPDC3 dysfunction

  • Targeting compensatory mechanisms in muscle cells

These approaches provide multiple avenues for therapeutic development, with the optimal strategy likely depending on the specific POPDC3 mutation and clinical presentation .

How can single-cell analysis enhance our understanding of POPDC3 function?

Single-cell analysis offers powerful approaches to enhance understanding of POPDC3 function:

• Single-cell RNA sequencing:

  • Reveals cell-type specific expression patterns within heterogeneous tissues

  • Identifies rare cell populations with distinctive POPDC3 expression

  • Maps transcriptional consequences of POPDC3 perturbation at cellular resolution

• Spatial transcriptomics:

  • Preserves tissue architecture while providing expression data

  • Maps POPDC3 expression to specific anatomical locations

  • Correlates with pathological features in disease states

• CyTOF/mass cytometry:

  • Simultaneously measures multiple protein markers with POPDC3

  • Quantifies signaling pathway activation states

  • Identifies cell subpopulations with distinct POPDC3 functions

• Live-cell imaging with fluorescent reporters:

  • Tracks POPDC3 localization in real-time

  • Monitors cAMP binding dynamics

  • Visualizes protein-protein interactions in living cells

These methodologies overcome limitations of bulk tissue analysis and capture the heterogeneity of POPDC3 function within complex tissues, potentially revealing new aspects of its biology in both normal physiology and disease states .

What experimental frameworks could address the relationship between POPDC3 and radioresistance mechanisms?

To investigate the relationship between POPDC3 and radioresistance mechanisms, the following experimental framework is recommended:

• In vitro radioresistance models:

  • Generate paired radioresistant/radiosensitive cancer cell lines

  • Manipulate POPDC3 expression using overexpression and knockdown approaches

  • Perform clonogenic survival assays after radiation with varying POPDC3 levels

• Mechanistic investigations:

  Mechanism	Experimental Approach	Outcome Measures
  DNA damage repair	Immunofluorescence for γH2AX foci	Quantification of repair kinetics
  Cell cycle checkpoints	Flow cytometry, Western blot	Cell cycle distribution, checkpoint activation
  Hypoxia response	HIF-1α reporter assays	Hypoxia pathway activation
  Cancer stem cells	Sphere formation assays	Stemness marker expression

• Signaling pathway analysis:

  • Phosphoproteomic analysis before and after radiation

  • Assessment of cAMP-dependent pathways

  • Investigation of MAPK activation (identified in GO analysis)

• In vivo validation:

  • Xenograft models with POPDC3 modulation

  • Fractionated radiation protocols

  • Tumor growth delay and local control assessments

• Clinical correlation:

  • Analysis of POPDC3 expression in pre- and post-radiation patient samples

  • Correlation with treatment outcomes and radiation response

This comprehensive framework addresses multiple aspects of radioresistance and provides a pathway from mechanistic understanding to potential clinical applications .

What quality control parameters are essential when working with recombinant POPDC3?

Essential quality control parameters for recombinant POPDC3 include:

• Purity assessment:

  • 90% purity via Bis-Tris PAGE or SDS-PAGE

  • Analytical SEC (HPLC) to detect aggregates

  • Mass spectrometry to confirm identity and modifications

• Functional validation:

  • cAMP binding assay to confirm Popeye domain activity

  • TREK-1 current modulation in heterologous expression systems

  • Proper membrane localization by immunofluorescence

• Structural integrity:

  • Circular dichroism to assess secondary structure

  • Thermal stability assays

  • Limited proteolysis to detect misfolding

• Endotoxin testing:

  • Essential for in vivo applications

  • Limulus amebocyte lysate (LAL) assay

• Storage stability:

  • Activity testing after storage under recommended conditions

  • Monitoring for degradation products

These quality control measures ensure experimental reproducibility and reliability of results when working with recombinant POPDC3 proteins .

How should researchers optimize immunodetection methods for POPDC3?

Optimizing immunodetection methods for POPDC3 requires careful consideration of several factors:

• Antibody selection:

  • Prefer antibodies targeting conserved epitopes in the Popeye domain

  • Validate specificity using POPDC3 knockout/knockdown controls

  • Consider using antibodies recognizing tags for recombinant proteins

• Western blot optimization:

  • Use mild detergents (0.5-1% Triton X-100 or NP-40) for extraction

  • Include protease inhibitors to prevent degradation

  • Optimize transfer conditions for this transmembrane protein (291 aa, ~33 kDa)

  • Block with 5% non-fat dry milk or BSA in TBST

• Immunohistochemistry/immunofluorescence:

  • Test multiple fixation methods (4% PFA, methanol)

  • Optimize antigen retrieval (citrate buffer, pH 6.0)

  • Include membrane permeabilization step

  • Use Tyramide Signal Amplification for low expression tissues

• Flow cytometry:

  • Gentle cell dissociation to preserve membrane proteins

  • Optimize permeabilization for intracellular epitopes

  • Include viability dye to exclude dead cells

• Controls:

  • Include tissue from POPDC3 knockout models as negative control

  • Use tissues with known high expression (skeletal muscle) as positive control

  • Include isotype control antibodies

These methodological considerations improve detection sensitivity and specificity for POPDC3 across various experimental applications .

What are the critical parameters for designing functional assays to assess POPDC3 activity?

When designing functional assays to assess POPDC3 activity, researchers should consider these critical parameters:

• cAMP binding assays:

  • Use purified recombinant POPDC3 with confirmed structural integrity

  • Employ [³H]-cAMP or fluorescent cAMP analogs

  • Include positive controls (other cAMP-binding proteins)

  • Determine binding kinetics (Kd, Bmax)

  • Compare wild-type with mutant POPDC3 variants

• TREK-1 current measurements:

  • Co-express POPDC3 and TREK-1 in Xenopus oocytes

  • Use two-electrode voltage clamp recordings

  • Measure current-voltage relationships

  • Assess modulation by varying cAMP concentrations

  • Compare wild-type with disease-associated mutants

• Membrane localization:

  • Generate fluorescent protein fusions

  • Confirm proper trafficking to plasma membrane

  • Quantify surface/internal distribution ratio

  • Use subcellular fractionation as complementary approach

• Protein-protein interaction assays:

  • Co-immunoprecipitation with potential partners

  • Proximity ligation assays in intact cells

  • FRET/BRET to measure interactions in living cells

  • Consider the effect of cAMP levels on interactions

These methodologically rigorous approaches allow for comprehensive assessment of POPDC3 functional activity in various experimental contexts and enable comparison between wild-type and mutant variants .

How can findings from POPDC3 research be integrated into personalized medicine approaches?

Integrating POPDC3 research into personalized medicine approaches offers several promising avenues:

• For muscular dystrophies:

  • Genetic testing for POPDC3 variants in undiagnosed LGMD patients

  • Phenotype-genotype correlations to predict disease progression

  • Potential therapy selection based on specific mutation mechanisms

  • Monitoring of muscle MRI patterns for early intervention

• For cancer management:

  • POPDC3 expression as a biomarker for radiotherapy response prediction

  • Integration into multiparameter nomograms for personalized prognosis

  • Potential radiation dose adjustment based on POPDC3 status

  • Combination therapy selection guided by POPDC3-associated pathways

• Clinical implementation considerations:

  • Development of standardized assays for clinical laboratories

  • Integration of POPDC3 testing into existing diagnostic algorithms

  • Prospective validation in diverse patient populations

  • Cost-effectiveness evaluation of testing strategies

This integrative approach bridges fundamental scientific discoveries with clinical applications, potentially improving diagnosis and treatment selection for both muscular dystrophies and cancers with aberrant POPDC3 function .

What interdisciplinary approaches would advance our understanding of POPDC3 biology?

Advancing POPDC3 biology requires interdisciplinary approaches integrating multiple scientific disciplines:

• Structural biology and biochemistry:

  • Cryo-EM or X-ray crystallography of POPDC3 alone and in complex with TREK-1

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Molecular dynamics simulations of cAMP binding and protein interactions

• Systems biology:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Mathematical modeling of cAMP signaling networks

  • Network analysis of POPDC3 interactome in different contexts

• Physiology and bioengineering:

  • Development of muscle-on-chip models with POPDC3 variants

  • Electrophysiological studies in engineered tissues

  • Biomechanical testing of muscle function

• Computational biology:

  • AI-driven predictions of mutation effects

  • Virtual screening for small molecule modulators

  • Mining of electronic health records for genotype-phenotype correlations

• Translational research:

  • Patient-derived organoids and iPSCs

  • Animal models with humanized POPDC3

  • Clinical biospecimen analysis with spatial resolution

This interdisciplinary framework leverages diverse expertise to address the complex biology of POPDC3 from molecular mechanisms to clinical implications .

What are the most significant knowledge gaps remaining in POPDC3 research?

Despite recent advances, significant knowledge gaps remain in POPDC3 research:

• Molecular mechanisms:

  • Complete interactome of POPDC3 beyond TREK-1

  • Structural basis of cAMP-induced conformational changes

  • Trafficking and turnover regulation

  • Post-translational modifications affecting function

• Physiological roles:

  • Precise function in muscle membrane integrity

  • Role in non-muscle tissues where it's expressed

  • Developmental functions versus adult maintenance

  • Interaction with other POPDC family members (functional redundancy)

• Pathological involvement:

  • Mechanism of radioresistance promotion in cancer

  • Potential role in other cancer types beyond HNSCC

  • Contribution to non-LGMD muscle disorders

  • Involvement in aging-related muscle changes

• Therapeutic targeting:

  • Druggable sites on POPDC3

  • Delivery methods for muscle-targeted therapies

  • Biomarkers of therapeutic response

  • Potential for compensatory approaches bypassing POPDC3