Recombinant Mouse Popeye domain-containing protein 3 (Popdc3)

What is the molecular structure of recombinant mouse POPDC3?

Recombinant full-length mouse POPDC3 protein consists of 291 amino acids (1-291aa) and is typically expressed with an N-terminal His-tag to facilitate purification. The protein contains a distinctive Popeye domain with a unique cAMP binding site, which is critical for its function . The amino acid sequence of mouse POPDC3 is:

MEKNSSLWKSLVTEHPLCTTWKQEAEGAIYHLASILFVVGFMGGSGFFGLLYVFSLLGLGFLSSAVWAWVDICAADIFSWNFVLFVICFMQFVHIAYQVHSITFARDFHVLYSSLFKPLGIPLPVFRTIALSSEVVSLEKEHCYAMQGKTSIDRLSVLISGRIRVTVDGEFLHYISPFQFLDSPEWDSLRPTEEGIFQVTLTADTDCRYVSWRRKKLYLLFAQHRYISRLFSVLIGSDIADKLYALNDRVYIGKKHHYDIRLPNYYHMSTPDLSRSPLTEQFRNSRQHCNK

This sequence contains transmembrane regions and the characteristic Popeye domain that enables binding to cyclic nucleotides, particularly cAMP.

What biological roles has POPDC3 been implicated in?

POPDC3 participates in multiple critical biological processes:

• Cell adhesion and motility, suggesting roles in tissue integrity and cell migration

• Membrane trafficking through interactions with other POPDC family proteins

• DNA methylation processes, indicating potential epigenetic regulatory functions

• Cancer progression, with reduced expression correlating with poor survival in gastric cancer patients

• Muscular function, as mutations are associated with limb-girdle muscular dystrophy (LGMD) type 26

The protein forms heteromeric complexes with other POPDC family members through a helix-helix interface at the C-terminus of the Popeye domain, which is essential for proper membrane localization and function .

How does recombinant mouse POPDC3 compare with human POPDC3?

While mouse and human POPDC3 share high sequence homology, species-specific differences may affect certain protein-protein interactions and functional details. Mouse models provide valuable insights, but researchers should be cautious when extrapolating findings directly to human systems.

What are the optimal conditions for reconstituting recombinant mouse POPDC3?

For successful reconstitution of lyophilized recombinant mouse POPDC3:

• Centrifuge the vial briefly 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% (optimally 50%) to prevent damage during freeze-thaw cycles

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C

• For working solutions, store aliquots at 4°C for up to one week

The protein is typically supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability during storage and reconstitution . Repeated freeze-thaw cycles should be strictly avoided as they can significantly reduce protein activity and integrity .

What techniques are most effective for studying POPDC3 protein-protein interactions?

Several methodologies have been validated for investigating POPDC3 interactions:

For BRET analysis specifically, researchers should test a range of expression ratios by varying the proportion of each plasmid during transfection while keeping the total amount constant, and measure 5 minutes after adding furimazine NanoLuc substrate .

What approaches can identify POPDC3's role in cancer progression?

To investigate POPDC3's involvement in cancer:

• Expression analysis:

  • Immunohistochemistry to compare POPDC3 levels between tumor and adjacent normal tissues (shown to be reduced in 85.71% of gastric tumors compared to normal mucosa)

  • Correlation of expression levels with patient survival and clinicopathological features

• Functional studies:

  • Knockdown/overexpression experiments to assess effects on cancer cell proliferation, migration, and invasion

  • Analysis of changes in DNA methylation patterns following POPDC3 modulation

• Radioresistance evaluation:

  • POPDC3 has been identified as a potential biomarker for radioresistance in head and neck squamous cell carcinoma (HNSCC)

  • Validation using databases such as Oncomine, Firebrowse, and Protein Atlas

• Pathway analysis:

  • Functional network enrichment using tools like DAVID to identify associated biological processes

  • Construction of protein-protein interaction networks using the STRING database and visualization with Cytoscape

How do POPDC3 mutations affect protein function and contribute to disease?

POPDC3 mutations can disrupt normal function through multiple mechanisms:

• Impaired membrane trafficking:

  • Certain mutations (particularly those affecting the C-terminal α-helices) disrupt the protein's ability to reach the cell membrane

  • Co-transfection experiments reveal that some disease-associated mutations (e.g., p.W188X) cause greater impairment of membrane trafficking

• Disrupted heteromeric complex formation:

  • Mutations affecting the helix-helix interface at the C-terminus of the Popeye domain can prevent normal interactions with other POPDC proteins

  • This interface contains ultra-conserved hydrophobic residues critical for proper function

• Disease mechanisms:

  • In limb-girdle muscular dystrophy (LGMD) type 26, POPDC3 gene variants lead to muscle weakness

  • Patient biopsy material shows only weakly diminished expression of POPDC1 and POPDC2, suggesting complex regulatory mechanisms

• Dual pathways of dysfunction:

  • Some mutations primarily affect membrane trafficking

  • Others leave protein-protein interactions intact but likely affect other essential functions

What are the key research gaps and future directions for POPDC3 research?

Despite recent advances, significant knowledge gaps remain:

• Molecular mechanisms:

  • Limited understanding of POPDC3's biological role, interacting proteins, downstream targets, and regulated signaling pathways

  • Unclear mechanisms connecting POPDC3 dysfunction to disease phenotypes

• Therapeutic potential:

  • Need for exploring POPDC3 as a therapeutic target in cancer

  • Developing strategies to correct or compensate for mutated POPDC3 in LGMD

• Structure-function relationships:

  • Detailed structural analysis of the Popeye domain and cAMP binding site

  • Understanding how cAMP binding modulates POPDC3 function and interactions

• Tissue-specific roles:

  • While highest expression occurs in skeletal muscle, POPDC3's functions in other tissues require further investigation

  • Potential tissue-specific interacting partners and signaling pathways

Future research directions should include comprehensive interactome studies, high-resolution structural analyses, development of animal models with tissue-specific POPDC3 modifications, and exploration of potential therapeutic approaches targeting POPDC3 or its downstream pathways.

What are common technical challenges when working with recombinant POPDC3?

Researchers frequently encounter several challenges when working with recombinant POPDC3:

• Protein stability issues:

  • As a transmembrane protein, POPDC3 may exhibit solubility and stability challenges

  • Store at -20°C/-80°C with proper aliquoting to prevent degradation

  • Avoid repeated freeze-thaw cycles which significantly reduce activity

• Expression and purification:

  • E. coli expression systems can yield high amounts of protein, but proper folding may be a concern

  • Purification typically achieves >90% purity as determined by SDS-PAGE

  • N-terminal His-tagging facilitates purification but may affect certain functional assays

• Buffer optimization:

  • Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been successfully used for storage

  • Addition of glycerol (5-50%) improves stability during storage

• Functional assays:

  • Demonstrating cAMP binding capacity requires specialized techniques

  • Protein-protein interaction studies require careful control experiments

  • Membrane trafficking assessments benefit from co-expression with other POPDC family members

How can POPDC3 expression analysis in tissues be optimized?

For reliable POPDC3 expression analysis:

• Sample preparation:

  • Fresh or properly preserved (flash-frozen) tissue samples yield most reliable results

  • For FFPE samples, optimize antigen retrieval methods for immunohistochemistry

• Detection methods:

  • Immunohistochemistry allows visualization of POPDC3 distribution in tissues

  • Western blotting provides semi-quantitative expression analysis

  • qRT-PCR offers sensitive mRNA quantification but should be complemented with protein analysis

• Controls and normalization:

  • Include appropriate positive controls (e.g., skeletal muscle) and negative controls

  • For cancer studies, analyze matched tumor and adjacent normal tissues from the same patient when possible

  • Use validated housekeeping genes or proteins for normalization

• Data interpretation:

  • Consider that reduced POPDC3 expression correlates with poor prognosis in gastric cancer

  • POPDC3 can serve as a potential biomarker for radioresistance in HNSCC

  • Integration with clinical parameters enhances prognostic value

How does POPDC3 compare functionally with other POPDC family members?

All three POPDC family members share the characteristic Popeye domain with cAMP binding capacity and form heteromeric complexes through helix-helix interfaces at the C-terminus of this domain . This complex formation is critical for proper membrane trafficking and localization . The presence of ultra-conserved hydrophobic residues in all family members highlights their functional importance in protein-protein interactions .

What bioinformatic approaches best support POPDC3 research?

Integrated bioinformatic strategies enhance POPDC3 research:

• Functional analysis:

  • Gene Ontology (GO) and KEGG pathway enrichment through the Database for Annotation, Visualization and Integrated Discovery (DAVID)

  • Identification of biological processes and pathways associated with POPDC3

• Interaction networks:

  • Construction of protein-protein interaction networks using the Search Tool for the Retrieval of Interacting Genes database (STRING)

  • Visualization and analysis of interaction networks with Cytoscape

• Expression validation:

  • Leveraging cancer databases like Oncomine, Firebrowse, and Protein Atlas to validate expression patterns

  • Correlation of expression with clinical parameters and outcomes

• Sequence analysis:

  • Identification of conserved domains and motifs

  • Prediction of post-translational modifications

  • Assessment of evolutionary conservation to identify functionally important regions

These approaches provide a comprehensive framework for understanding POPDC3's biological context, identifying potential interacting partners, and generating hypotheses for experimental validation.