Recombinant Chicken DnaJ homolog subfamily C member 3 (DNAJC3)

What is the structure and function of chicken DNAJC3?

Chicken DNAJC3 is a co-chaperone protein that works with BiP (immunoglobulin heavy-chain binding protein) to facilitate proper protein folding in the endoplasmic reticulum. Structurally, it contains J-domains that regulate BiP's ATPase activity and subsequent substrate binding. The protein is expressed ubiquitously but shows particularly high expression in pancreatic tissue including β-cells and in hepatocytes .

Functionally, DNAJC3 serves as an inhibitor of PKR (protein kinase RNA-activated) and PERK (PKR-like ER kinase), thereby attenuating ER stress responses. This inhibition prevents excessive cell apoptosis under ER stress conditions, which is particularly crucial for pancreatic β-cell survival. Loss of DNAJC3 function leads to activation of cellular apoptosis pathways, resulting in β-cell loss and decreased insulin secretion, as demonstrated in knockout models .

How does chicken DNAJC3 differ from mammalian DNAJC3 homologs?

While chicken and mammalian DNAJC3 share conserved functional domains, species-specific differences exist in amino acid sequences and post-translational modifications. These differences may result in subtle functional variations in how the protein responds to ER stress across species. Comparative sequence analysis shows conservation of key functional domains, particularly the J-domain, which is essential for interaction with BiP.

When expressing recombinant chicken DNAJC3 in heterologous systems, researchers should consider these species-specific differences, as they may affect protein folding, activity, and interaction with experimental systems. Sequence alignment studies have shown approximately 85-90% homology between chicken and human DNAJC3 in critical functional domains, suggesting conserved core functions across species while allowing for species-specific adaptations.

What expression systems are most effective for producing recombinant chicken DNAJC3?

For functional studies requiring properly folded and modified DNAJC3, eukaryotic expression systems such as insect cells (Sf9, Sf21) using baculovirus vectors or mammalian cells (HEK293, CHO) are preferred. These systems provide appropriate post-translational modifications and folding environments. The choice of expression system should align with downstream applications:

When using bacterial systems, solubility can be improved by expressing DNAJC3 as a fusion protein with tags such as MBP (maltose-binding protein) or using specialized E. coli strains designed for disulfide bond formation.

How does recombinant chicken DNAJC3 interact with the unfolded protein response pathway in avian cells?

The interaction between recombinant chicken DNAJC3 and the unfolded protein response (UPR) pathway in avian cells involves complex regulatory mechanisms. DNAJC3 functions as a negative regulator of PERK signaling, a critical branch of the UPR. Under normal conditions, DNAJC3 helps maintain ER homeostasis by preventing hyperactivation of stress responses.

When investigating these interactions experimentally, researchers should consider establishing avian cell models with controlled DNAJC3 expression. Techniques such as RNAi-mediated knockdown or CRISPR-Cas9 genome editing followed by rescue with recombinant DNAJC3 can elucidate specific roles. Key UPR markers to monitor include:

• Phosphorylation status of eIF2α

• Expression levels of ATF4, CHOP, and XBP1

• Calcium signaling dynamics

• Activation of pro-apoptotic pathways

Comparative studies between mammalian and avian cells have revealed both conserved and divergent aspects of DNAJC3 function in UPR regulation. The avian-specific aspects of this regulation may provide insights into species-specific adaptations to ER stress, particularly relevant for understanding avian metabolic disorders and immune responses.

What role does DNAJC3 play in chicken primordial germ cell (PGC) DNA repair pathways?

Emerging research suggests potential involvement of DNAJC3 in DNA repair mechanisms in chicken primordial germ cells (PGCs). While direct evidence is limited, the observed elevation of base excision repair (BER) pathway genes in chicken PGCs indicates a complex interplay between ER stress responses and DNA repair mechanisms .

When investigating DNAJC3's potential role in these pathways, researchers should consider:

• The expression correlation between DNAJC3 and key BER proteins such as uracil N-glycosylase (UNG)

• Changes in BER efficiency following DNAJC3 modulation

• Potential direct interactions between DNAJC3 and DNA repair complexes

Recent findings indicate that chicken PGCs exhibit distinctly elevated BER pathway gene expression compared to somatic cells like DF-1 fibroblasts, which affects genome editing efficiency . This suggests PGCs have unique DNA repair properties that may involve ER stress response proteins like DNAJC3.

To explore this connection, co-immunoprecipitation assays with recombinant chicken DNAJC3 and key BER proteins, followed by mass spectrometry, could identify novel protein-protein interactions. Additionally, investigating DNAJC3 knockdown effects on DNA repair efficiency in PGCs could provide functional evidence for its role in maintaining genomic integrity.

How does lipid metabolism affect DNAJC3 function in chicken cells, and what experimental approaches best characterize this relationship?

The relationship between lipid metabolism and DNAJC3 function represents an emerging area of research with significant implications for understanding metabolic disorders. Proteomic studies in human DNAJC3-deficient cells have revealed dysregulation of lipid homeostasis markers, including upregulation of SOAT1 (Sterol O-acyltransferase 1) and PLIN2 (perilipin-2) .

To investigate this relationship in chicken cells, researchers should employ a multi-omics approach:

• Lipidomics analysis to profile changes in lipid composition following DNAJC3 modulation

• Transcriptomics to identify altered expression of lipid metabolism genes

• Metabolic flux analysis using isotope-labeled lipid precursors

Experimental approaches should include:

Recent findings indicate that loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and mitochondrial dysfunction . In chicken cells, investigating whether similar mechanisms operate could reveal conserved pathways of DNAJC3-mediated protection against lipotoxicity.

What purification strategies yield the highest activity for recombinant chicken DNAJC3?

Purification of recombinant chicken DNAJC3 with optimal activity requires careful consideration of expression systems, buffer conditions, and purification techniques. The following strategy has been optimized based on protein characteristics:

For bacterial expression:

• Express DNAJC3 with an N-terminal His-tag in E. coli BL21(DE3) using pET expression systems

• Induce expression at low temperature (16-18°C) overnight with 0.1-0.5 mM IPTG to enhance proper folding

• Use lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Employ a two-step purification process:

  • Initial Ni-NTA affinity chromatography

  • Secondary size exclusion chromatography using Superdex 200

Critical factors affecting DNAJC3 activity include:

Activity assays should measure DNAJC3's ability to stimulate the ATPase activity of recombinant BiP, which provides a functional readout of co-chaperone activity. Circular dichroism spectroscopy can confirm proper folding, while thermal shift assays help optimize buffer conditions for maximum stability.

How can researchers effectively design experiments to study the interaction between recombinant chicken DNAJC3 and BiP?

Studying the interaction between recombinant chicken DNAJC3 and BiP requires a comprehensive experimental approach that combines biochemical, biophysical, and cellular techniques:

• In vitro binding assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics and affinity constants

  • Isothermal Titration Calorimetry (ITC) to measure thermodynamic parameters

  • Microscale Thermophoresis (MST) for detecting interactions in solution

• Structural studies:

  • Co-crystallization of DNAJC3-BiP complexes for X-ray crystallography

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

  • Cross-linking mass spectrometry to identify proximity of specific residues

• Functional assays:

  • ATPase activity measurements to quantify BiP stimulation by DNAJC3

  • Protein aggregation assays to assess chaperone activity modulation

  • FRET-based assays to monitor conformational changes during interaction

When designing mutation studies, focus on conserved residues in the J-domain, particularly the HPD motif that is critical for stimulating BiP's ATPase activity. Consider creating a panel of chicken DNAJC3 variants with point mutations in key domains to map the functional importance of specific regions.

For cellular assays, develop systems that allow visualization of these interactions in living cells using techniques such as:

• Split-GFP complementation

• Proximity ligation assay (PLA)

• Förster resonance energy transfer (FRET) with fluorescently tagged proteins

These approaches provide complementary data on both physical interactions and functional consequences of DNAJC3-BiP binding.

What are the best methods for evaluating the protective effects of recombinant chicken DNAJC3 against ER stress in avian cell models?

To evaluate the protective effects of recombinant chicken DNAJC3 against ER stress in avian cell models, researchers should implement a multi-faceted experimental approach:

• Cell models and stress induction:

  • Establish avian cell lines with modulated DNAJC3 expression (overexpression, knockdown, knockout)

  • Use primary chicken pancreatic β-cells when possible for physiological relevance

  • Induce ER stress using diverse stressors (tunicamycin, thapsigargin, brefeldin A, palmitate) to activate different UPR branches

• ER stress markers to monitor:

• Functional outcomes assessment:

  • Cell viability assays (MTT, XTT, ATP content)

  • Apoptosis measurements (Annexin V/PI staining, caspase activity)

  • ER morphology visualization (transmission electron microscopy)

  • Calcium homeostasis (fluorescent calcium indicators)

  • Protein synthesis rates (puromycin incorporation)

• Time-course experiments:
  Design experiments to capture both acute (0-24h) and chronic (24-72h) effects of DNAJC3 protection, as different mechanisms may predominate at different time points.

• Recovery experiments:
  Assess the capacity of cells to recover from ER stress upon removal of stressors, comparing recovery kinetics between cells with normal versus altered DNAJC3 expression.

When delivering recombinant DNAJC3 to cells, consider protein transduction methods using cell-penetrating peptides or nanoparticle-based delivery systems to achieve physiologically relevant intracellular concentrations.

How should researchers analyze proteomics data to identify pathways affected by DNAJC3 deficiency in chicken models?

Analyzing proteomics data to identify pathways affected by DNAJC3 deficiency requires a structured analytical approach:

• Initial data processing:

  • Apply appropriate normalization methods (e.g., total ion current, LOESS)

  • Implement robust statistical tests for differential expression analysis (ANOVA with multiple testing correction)

  • Filter proteins based on fold change (typically ≥1.5-fold) and significance (p<0.05)

• Pathway analysis workflow:

  • Perform Gene Ontology (GO) enrichment analysis for biological processes, molecular functions, and cellular components

  • Conduct KEGG, Reactome, and DAVID pathway mapping to identify enriched functional categories

  • Apply network analysis using STRING, Cytoscape, or IPA to visualize protein-protein interactions

• Key pathways to focus on:
  Based on human DNAJC3 deficiency studies, pay particular attention to:

  • ER stress response and unfolded protein response

  • Lipid metabolism and cholesterol homeostasis pathways

  • Mitochondrial function and energy metabolism

  • Protein synthesis and degradation pathways

  • Cell death and survival mechanisms

A recent proteomics study of human DNAJC3-deficient fibroblasts identified 24 significantly altered proteins (12 upregulated, 12 downregulated), with notable changes in proteins involved in lipid metabolism (SOAT1, PLIN2), mitochondrial function (SDHB, APOOL, ACADSB), and vesicular transport (GGA1, CHMP6) . For chicken models, similar pathway alterations may be expected, potentially with avian-specific variations.

Integrating proteomic findings with transcriptomics and metabolomics data using multi-omics approaches can provide a more comprehensive view of the biological impact of DNAJC3 deficiency.

What statistical approaches are most appropriate for analyzing the efficacy of recombinant DNAJC3 in protection against ER stress-induced apoptosis?

When analyzing the efficacy of recombinant DNAJC3 in protection against ER stress-induced apoptosis, researchers should employ robust statistical approaches tailored to the experimental design:

• Experimental design considerations:

  • Include appropriate sample sizes (power analysis recommended)

  • Implement technical and biological replicates

  • Use factorial designs to test multiple conditions (DNAJC3 concentrations × stress durations × stress types)

• Statistical methods by data type:

How can researchers effectively compare the functional differences between wild-type and mutant forms of chicken DNAJC3?

To effectively compare functional differences between wild-type and mutant forms of chicken DNAJC3, researchers should implement a comprehensive analytical framework that combines in vitro, cellular, and computational approaches:

• Structural and biophysical comparison:

  • Circular dichroism (CD) spectroscopy to assess secondary structure differences

  • Thermal shift assays to determine protein stability

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to evaluate oligomerization state

  • Hydrogen-deuterium exchange mass spectrometry to map conformational differences

• Functional assays:

  • BiP ATPase stimulation activity using colorimetric phosphate release assays

  • Protein aggregation prevention assays using model substrates

  • Client protein binding affinity measurements

  • Co-immunoprecipitation efficiency with interaction partners

• Cellular assays:

  • Complementation studies in DNAJC3-knockout cells

  • ER stress resistance measurement after reconstitution

  • Subcellular localization analysis using confocal microscopy

  • Protein half-life determination using cycloheximide chase assays

• Data integration and visualization:
  Create comprehensive comparison tables with quantitative metrics:

• Structure-function correlations:

  • Map mutational effects to specific domains and motifs

  • Use molecular dynamics simulations to predict functional consequences

  • Develop predictive models correlating structural changes with functional outcomes

For comprehensive assessment, design mutations targeting different functional domains (J-domain, substrate binding region, BiP interaction sites) to create a detailed map of structure-function relationships in chicken DNAJC3.

What are common challenges in expressing and purifying recombinant chicken DNAJC3, and how can researchers overcome them?

Expressing and purifying recombinant chicken DNAJC3 presents several technical challenges that researchers commonly encounter. This systematic troubleshooting guide addresses key issues and their solutions:

• Low expression yields in bacterial systems:

• Protein instability during purification:

  • Include protease inhibitors (PMSF, EDTA-free cocktail) in all buffers

  • Add reducing agents (5mM DTT or 10mM β-mercaptoethanol) to prevent oxidation

  • Maintain constant cold temperature (4°C) throughout purification

  • Consider adding 10% glycerol to all buffers to enhance stability

• Limited purity after initial chromatography:

  • Implement a multi-step purification strategy:
    a. IMAC (Ni-NTA) for initial capture
    b. Ion exchange chromatography as intermediate step
    c. Size exclusion chromatography as polishing step

  • Optimize imidazole concentration gradients (typically 20-500mM) to reduce non-specific binding

• Loss of functional activity:

  • Validate protein folding by circular dichroism spectroscopy

  • Test buffer conditions with thermal shift assays to identify stabilizing additives

  • Consider protein engineering approaches to enhance stability

  • Avoid freeze-thaw cycles; store purified protein as single-use aliquots

• Heterogeneity in post-translational modifications:

  • For applications requiring homogeneous protein, express in bacterial systems and focus on core functional domains

  • For studies of native-like modifications, use avian cell lines despite potentially lower yields

When expressing difficult constructs, consider split domain approaches, where individual domains are expressed and purified separately, then used for domain-specific functional studies.

How can researchers address conflicting results when comparing in vitro versus cellular effects of recombinant DNAJC3?

Addressing conflicting results between in vitro and cellular studies of recombinant DNAJC3 requires systematic investigation of potential sources of discrepancy:

• Systematic analysis framework:

  • Document all differences between experimental systems (protein concentration, buffer components, cell types, assay conditions)

  • Implement bridging experiments that gradually transition from in vitro to cellular conditions

  • Validate findings using complementary methodologies

• Common sources of discrepancy and solutions:

• Technical approaches to resolve conflicts:

  • Use semi-permeabilized cell systems as intermediate between purified and cellular contexts

  • Employ microinjection of recombinant proteins to bypass delivery barriers

  • Develop reconstituted membrane systems that better mimic the ER environment

  • Create DNAJC3-knockout cellular backgrounds for cleaner complementation studies

• Data interpretation strategies:

  • Consider that both in vitro and cellular observations may be correct within their contexts

  • Develop integrated models that reconcile apparent contradictions

  • Identify condition-dependent factors that switch DNAJC3 between different functional modes

• Case study example:
  A common discrepancy is the protective effect of DNAJC3 against ER stress. In vitro assays may show direct inhibition of PERK phosphorylation, while cellular studies might show complex time-dependent effects due to feedback loops. Resolving this requires time-resolved studies in both systems and consideration of network effects present only in cellular contexts.

Remember that discrepancies often reveal important biological insights about context-dependent regulation and function that neither system alone could identify.

What strategies can researchers use to ensure reproducibility in studies comparing wild-type and mutant DNAJC3 proteins?

Ensuring reproducibility in comparative studies of wild-type and mutant chicken DNAJC3 proteins requires rigorous attention to experimental design, execution, and reporting:

• Protein preparation standardization:

  • Use identical expression systems, purification protocols, and buffer conditions

  • Characterize all protein preparations using multiple quality control methods:

    • SDS-PAGE for purity assessment

    • Western blot for identity confirmation

    • Dynamic light scattering for aggregation analysis

    • Circular dichroism for secondary structure verification

  • Prepare large, homogeneous batches and store as single-use aliquots

• Experimental design considerations:

  • Implement randomization and blinding where possible

  • Include technical replicates (same protein preparation) and biological replicates (independent preparations)

  • Determine appropriate sample sizes through power analysis

  • Use factorial designs to test interactions between variables

• Controls and validation:

  • Include positive and negative controls in all experiments

  • Verify functionality using multiple orthogonal assays

  • Validate key findings using different experimental approaches

  • For cellular studies, use multiple cell lines or primary cells

• Comprehensive documentation:
  Create detailed protocols capturing:

  • Exact buffer compositions (including lot numbers of key components)

  • Incubation times and temperatures

  • Instrument settings and calibration status

  • Raw data processing methods with version-controlled scripts

• Reporting standards:
  Follow best practices for reporting:

  • Provide complete methods enabling reproduction

  • Share raw data in public repositories

  • Report all statistical analyses comprehensively

  • Disclose any experimental attempts that failed

• Reproducibility checklist:

• Specific recommendations for DNAJC3 studies:

  • Include stability assessments before each experiment, as DNAJC3 may be prone to time-dependent activity loss

  • Test for concentration-dependent effects, as DNAJC3 function may vary with concentration

  • Account for potential oxidation sensitivity by measuring and reporting redox conditions

By implementing these strategies, researchers can enhance the reliability and reproducibility of their comparative DNAJC3 studies, contributing to a more robust scientific understanding of this important protein's function.