Recombinant Chicken 14-3-3 protein zeta (YWHAZ)

What is the basic structure of chicken 14-3-3 protein zeta (YWHAZ)?

Chicken YWHAZ consists of 245 amino acids with a molecular weight of approximately 31.13 kDa and a theoretical isoelectric point of 4.82. The protein forms a homodimer as its functional unit and has a highly conserved structure compared to mammalian homologs. The full sequence is: MDKNELVQKA KLAEQAERYD DMASCMKSVT EQGAELSNEE RNLLSVAYKN VVGARRSSWR VVSSIEQKTE GAEKKQQMAR EYREKIETEL RDICNDVLSL LEKFLIPNAS QAESKVFYLK MKGDYYRYLA EVAAGDDKKG IVEQSQQAYQ EAFEISKKEM QPTHPIRLGL ALNFSVFYYE ILNSPEKACS LAKTAFDEAI AELDTLSEES YKDSTLIMQL LRDNLTLWTS DTQGDEAEAG EGGEN . Bioinformatic analysis shows high sequence conservation with 14-3-3 proteins from other species, sharing over 99% amino acid sequence identity with homologs from E. stiedae and E. magna .

What cellular functions does chicken YWHAZ participate in?

Chicken YWHAZ functions as an adapter protein implicated in regulating a large spectrum of both general and specialized signaling pathways. It binds to numerous partner proteins, typically recognizing phosphoserine or phosphothreonine motifs. This binding generally results in modulation of the binding partner's activity . YWHAZ is involved in crucial cellular processes including signal transduction, cell cycle regulation, and apoptosis . Research has demonstrated that 14-3-3σ (another member of the 14-3-3 family related to YWHAZ) can inhibit cell proliferation and cell cycle progression by regulating CDK2/CDC2/p53 expressions in certain cellular contexts .

How conserved is chicken YWHAZ compared to mammalian homologs?

Chicken YWHAZ shows remarkable conservation with mammalian 14-3-3 proteins. Studies using specific antibodies against mammalian isoforms to probe for 14-3-3 isoforms in adult hen brains suggested a high degree of similarity in primary structure, at least in regions containing the epitopes. Reverse-phase HPLC analysis of purified avian 14-3-3 proteins indicates similarity in sequence and levels of expression comparable to mammalian counterparts . This conservation explains why chicken YWHAZ can be used as a model for studying 14-3-3 protein function across species.

What expression systems are recommended for recombinant chicken YWHAZ production?

Several expression systems have been successfully used for producing recombinant YWHAZ:

• Yeast expression system: Used for producing chicken YWHAZ (AA 1-245) with His tag, offering good protein yields with proper folding .

• E. coli expression system: Commonly used for human YWHAZ expression with >95% purity; adaptable for chicken YWHAZ with appropriate codon optimization .

• Baculovirus/insect cell system: Though more complex, this system can provide higher-quality protein with proper post-translational modifications .

The choice depends on your specific requirements. For structural studies requiring high purity, E. coli may be sufficient. For functional studies where post-translational modifications are important, yeast or insect cell systems are preferable. When expressing the protein, ensure the construct contains the full coding sequence (816 bp for chicken YWHAZ) and appropriate purification tags.

What challenges might researchers encounter when purifying recombinant chicken YWHAZ?

Common challenges in purifying recombinant chicken YWHAZ include:

• Protein solubility issues: 14-3-3 proteins can form inclusion bodies in bacterial systems. Solution: Optimize induction conditions (lower temperature, reduced IPTG concentration) or use fusion tags like GST that enhance solubility.

• Maintaining dimer stability: YWHAZ functions as a homodimer, and purification conditions can affect dimer formation. Solution: Avoid harsh elution conditions and include stabilizing agents like glycerol in purification buffers.

• Protein-protein interactions: YWHAZ naturally binds many partners, which can co-purify. Solution: Use high salt concentrations (300-500 mM NaCl) in washing steps and consider ion exchange chromatography as a second purification step after affinity purification.

• Maintaining protein activity: Preserving phosphopeptide binding capability. Solution: Include reducing agents like DTT (0.25-1 mM) in storage buffers and avoid multiple freeze-thaw cycles.

An effective purification buffer system contains 50 mM sodium phosphate, pH 7.0, 300 mM NaCl, 0.25 mM DTT, and 25% glycerol for storage stability .

What are the optimal methods for detecting and quantifying chicken YWHAZ in biological samples?

Several methods are effective for detecting chicken YWHAZ in biological samples:

• ELISA: Commercial sandwich ELISA kits offer high sensitivity and specificity for chicken YWHAZ detection in serum, plasma, tissue homogenates, and cell culture supernatants. These kits typically have detection ranges in the ng/mL range with intra-CV and inter-CV values provided with the kit .

• Western Blotting: Effective for semi-quantitative detection using either anti-YWHAZ antibodies or anti-tag antibodies if working with recombinant tagged protein. Recommended loading controls include housekeeping proteins like GAPDH or β-actin.

• qPCR: For measuring YWHAZ mRNA expression levels. When used as a reference gene, careful validation is necessary as YWHAZ expression can change under certain conditions. The threshold setting for qPCR should be in the log-linear phase of amplification .

• Immunohistochemistry: Useful for localizing YWHAZ in tissue sections and determining subcellular localization.

For quantitative comparisons across multiple samples, ELISA provides the most reliable results with minimal variability (reported standard deviation <8% when the same standard is repeated 20 times on the same plate) .

How can researchers verify the functionality of recombinant chicken YWHAZ after purification?

To verify functionality of recombinant chicken YWHAZ, consider these methodological approaches:

• Phosphopeptide binding assay: YWHAZ binds phosphorylated client proteins. Use fluorescence polarization or isothermal titration calorimetry with known phosphopeptides (e.g., from Raf-1) to verify binding activity .

• Protein-protein interaction assays: Co-immunoprecipitation or pull-down assays with known binding partners can confirm functional binding activity.

• Structural verification:

  • Circular dichroism to ensure proper secondary structure

  • Size exclusion chromatography to confirm dimer formation

  • Limited proteolysis to verify proper folding

• Cellular assays: If testing in a cellular context, overexpression or addition of purified YWHAZ should induce expected phenotypes, such as effects on cell cycle progression or signal transduction pathways .

A key functional test is the effect on cell cycle regulation in an appropriate cellular model, as 14-3-3 proteins regulate cell cycle by interacting with proteins like CDK2 and CDC2 .

How should researchers design experiments to investigate chicken YWHAZ function in cellular processes?

A comprehensive experimental approach should include:

• Expression modulation strategies:

  • Overexpression: Clone the full chicken YWHAZ gene into an appropriate expression vector (e.g., pcDNA3.1) for transfection into relevant cell lines

  • Knockdown: Design siRNAs targeting chicken YWHAZ. Example target sequence: 5′-CCCUCCAGGCCGAGCGCUGGC-3′

  • CRISPR/Cas9 knockout: Consider engineering specific mutations, such as the 7-bp deletion (380_387delCCTGGCA) used in zebrafish models

• Phenotypic readouts:

  • Cell proliferation assays (MTT, BrdU incorporation)

  • Cell cycle analysis by flow cytometry

  • Apoptosis assays (Annexin V staining, caspase activity)

  • Cell migration/invasion assays

• Molecular mechanism investigation:

  • RNA-seq or proteomic analysis to identify affected pathways

  • ChIP-seq for transcription factor interactions

  • Co-immunoprecipitation to identify binding partners

  • Western blotting to measure expression changes in key signaling molecules (p53, CDK2, CDC2)

• Validation in relevant model systems:

  • Primary chicken cell cultures

  • Ex vivo tissue explants

  • In vivo models where appropriate

For cell cycle studies specifically, researchers should include both cell proliferation assays and flow cytometry analysis to determine the distribution of cells in different cell cycle phases (G0/G1, S, G2/M) .

What controls are essential when studying recombinant chicken YWHAZ in experimental systems?

Essential controls for YWHAZ experiments include:

• Expression controls:

  • Empty vector transfection control for overexpression studies

  • Non-targeting siRNA/shRNA for knockdown studies

  • Wild-type cells for CRISPR experiments

• Protein function controls:

  • Mutant versions of YWHAZ (e.g., phosphobinding site mutations)

  • Other 14-3-3 isoforms to test specificity (e.g., YWHAQ/14-3-3 theta)

  • Rescue experiments where the knockdown/knockout phenotype is complemented with recombinant YWHAZ expression

• Technical controls:

  • Positive and negative controls for protein-protein interaction assays

  • Multiple housekeeping genes for normalization in qPCR (GAPDH, β-actin)

  • Loading controls for Western blots

  • Isotype controls for immunostaining

• Experimental design controls:

  • Biological replicates (minimum n=3)

  • Technical replicates for each measurement

  • Time-course studies when investigating dynamic processes

When studying chicken YWHAZ overexpression effects, it's crucial to verify expression levels by both qPCR and Western blot and to compare multiple expression levels to avoid artifacts from extreme overexpression .

How is chicken YWHAZ used in studying viral pathogenesis and oncogenic mechanisms?

Chicken YWHAZ has proven valuable for studying disease mechanisms, particularly in avian leukosis virus (ALV) research:

• ALV-J-induced fibrosarcoma models:

  • Studies have shown that ALV-J-SD1005 strain infection leads to decreased chicken 14-3-3σ expression and increased cell proliferation in DF-1 cells

  • Chicken 14-3-3σ overexpression significantly decreased cell proliferation and S-phase cells while increasing G2/M-phase cells in ALV-J-infected cells

  • This regulation occurs through modulation of CDK2/CDC2/p53 expression

• Investigation methodology:

  • Viral inoculation of cells with 10² TCID₅₀ of ALV-J-SD1005 strain

  • Analysis of cell proliferation via MTT assay or direct cell counting

  • Flow cytometry for cell cycle analysis

  • qPCR and Western blot analysis of 14-3-3σ, CDK2, CDC2, and p53 expression

• YWHAZ as a potential therapeutic target:

  • Based on its ability to inhibit cell proliferation, YWHAZ could be targeted to control virus-induced pathological cellular proliferation

The relationship between viral infection, 14-3-3σ expression, and cell proliferation provides insights into mechanisms that could be relevant to human cancer biology as well .

What role does YWHAZ play in cancer biology and how can chicken YWHAZ research inform human cancer studies?

YWHAZ has significant implications in cancer biology, and chicken YWHAZ research provides valuable models:

• Comparative oncology insights:

  • YWHAZ overexpression has been linked to tumor cell proliferation in multiple human cancers including gastric, bladder, and prostate cancers

  • Chicken models allow investigation of conserved mechanisms in a controlled system

• Prognostic significance:

  • In human studies, YWHAZ positivity correlates with worse clinical outcomes and higher recurrence rates

  • For example, in gastric cancer, YWHAZ overexpression significantly correlated with larger tumor size, venous and lymphatic invasion, deeper tumor depth, and higher pathological stage

• Mechanistic insights:

  • YWHAZ knockdown inhibits proliferation, migration, and invasion in cancer cells

  • In ALV-J infection models, 14-3-3σ was found to negatively regulate cell proliferation similarly to mammalian systems

  • YWHAZ may contribute to genomic instability in cancers, with links to alterations in genes like MYC, PALB2, TP53, and CXCL2

• Translational potential:

  • Studies in chicken systems can identify potential therapeutic targets conserved in humans

  • Approaches successful in modulating chicken YWHAZ function may inform human cancer therapeutic strategies

Research suggests YWHAZ could be both a prognostic biomarker and potential therapeutic target in various cancers, with hazard ratios of 2.3 (95% CI: 1.003–5.304) reported in some human cancer studies .

How can researchers utilize protein-protein interaction studies to understand chicken YWHAZ function?

Researchers can employ several sophisticated approaches to study YWHAZ protein-protein interactions:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use specific antibodies against chicken YWHAZ or epitope tags in recombinant constructs

  • Consider crosslinking approaches for transient interactions

  • Analyze by mass spectrometry to identify novel binding partners

  • Verify interactions by reciprocal Co-IP

• Yeast two-hybrid screening:

  • Use chicken YWHAZ as bait to screen chicken cDNA libraries

  • Validate interactions in mammalian systems with techniques like FRET or BiFC

  • Consider split-ubiquitin systems for membrane protein interactions

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proximal proteins in living cells

  • APEX2 fusions for rapid labeling of neighboring proteins

• Structural studies:

  • X-ray crystallography of YWHAZ with phosphopeptides from binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cryo-EM for larger complexes

• Modulators of interactions:

  • Small molecule compounds like compound 15 (which targets the phosphopeptide binding site)

  • Peptide mimetics of known binding interfaces

When studying YWHAZ interactions with specific partners like CDK2 or CDC2, researchers should focus on phosphorylation-dependent interactions, as 14-3-3 proteins typically bind phosphorylated motifs on partner proteins .

What advanced genomic approaches are recommended for studying YWHAZ regulation in chicken models?

Advanced genomic approaches for studying chicken YWHAZ regulation include:

• Transcriptional regulation analysis:

  • ChIP-seq to identify transcription factors binding to the YWHAZ promoter

  • ATAC-seq to assess chromatin accessibility at the YWHAZ locus

  • CUT&RUN for higher resolution transcription factor binding profiles

  • Reporter assays with serial deletions of the promoter to identify regulatory elements

• Post-transcriptional regulation:

  • RIP-seq to identify RNA-binding proteins interacting with YWHAZ mRNA

  • miRNA profiling and target validation (e.g., miR-375 has been associated with YWHAZ regulation in some contexts)

  • RNA stability assays following actinomycin D treatment

• Epigenetic regulation:

  • Bisulfite sequencing to assess DNA methylation patterns

  • ChIP-seq for histone modifications at the YWHAZ locus

  • 3D chromatin organization (Hi-C) to identify distant regulatory elements

• Functional genomics:

  • CRISPR interference or activation to modulate YWHAZ expression

  • CRISPR tiling screens to identify functional regulatory elements

  • Allele-specific expression analysis in hybrid models

• Systems biology approaches:

  • Integration of multi-omics data (RNA-seq, proteomics, metabolomics)

  • Principal component analysis to characterize expression patterns

  • Network analysis to place YWHAZ in broader signaling contexts

When analyzing data, consider nested experimental designs and appropriate statistical approaches for qPCR data analysis, including proper selection of reference genes and threshold setting in the log-linear phase of amplification curves .

What are common pitfalls in chicken YWHAZ overexpression studies and how can they be avoided?

Common pitfalls and solutions in YWHAZ overexpression studies include:

• Non-physiological expression levels:

  • Pitfall: Extremely high expression can cause artificial phenotypes not relevant to normal function

  • Solution: Use inducible expression systems or weak promoters to achieve near-physiological levels; verify expression against endogenous levels

• Improper controls:

  • Pitfall: Using empty vectors that don't account for the metabolic burden of protein expression

  • Solution: Include controls expressing unrelated proteins of similar size; use mutant versions of YWHAZ as functional controls

• Interference with endogenous protein:

  • Pitfall: Tagged YWHAZ may not interact properly with endogenous partners

  • Solution: Test multiple tag positions (N-terminal, C-terminal, internal); verify key interactions; use smaller tags

• Cell line variability:

  • Pitfall: Different cell lines respond differently to YWHAZ overexpression

  • Solution: Test effects in multiple cell lines; include primary cells where possible; consider species-specific context

• Timing issues:

  • Pitfall: Missing temporal dynamics of YWHAZ effects

  • Solution: Perform time-course studies; use inducible systems to capture immediate vs. long-term effects

In ALV-J infection studies, researchers successfully employed pcDNA3.1-14-3-3σ plasmids for transfection using Lipofectamine 3000 reagent. They collected cells at 24 hours post-transfection for optimal expression analysis .

How can researchers address inconsistent results when studying YWHAZ interactions with binding partners?

Addressing inconsistent results in YWHAZ interaction studies:

• Phosphorylation status:

  • Issue: YWHAZ binds phosphorylated motifs, but phosphorylation status can vary with conditions

  • Solution: Use phosphatase inhibitors consistently; consider phosphomimetic mutations in partners; verify phosphorylation status of binding partners

• Buffer conditions affecting interactions:

  • Issue: Salt concentration, pH, and detergents can disrupt interactions

  • Solution: Standardize buffer conditions; test multiple conditions; use crosslinking approaches for transient interactions

• Cell type-specific cofactors:

  • Issue: Different cell types may have different cofactors affecting YWHAZ interactions

  • Solution: Test interactions in multiple cell types; consider pull-downs with purified components

• Isoform confusion:

  • Issue: Antibodies may cross-react between 14-3-3 isoforms

  • Solution: Verify antibody specificity; use tagged versions; consider isoform-specific knockdown

• Competition between binding partners:

  • Issue: YWHAZ has multiple binding partners that may compete for binding

  • Solution: Consider cellular context; use in vitro systems with purified components; develop quantitative binding assays

• Technical approaches:

  • Issue: Different techniques may yield different results

  • Solution: Verify key interactions with multiple techniques (Co-IP, proximity labeling, FRET, etc.)

Studies on modulators of 14-3-3 protein-protein interactions have shown that binding energy changes with mutations in binding interfaces, highlighting the sensitivity of these interactions to structural changes .

What are emerging approaches for studying chicken YWHAZ in neurodevelopmental and immunological contexts?

Emerging approaches in YWHAZ research span multiple fields:

• Neurodevelopmental applications:

  • Whole-brain imaging techniques to study YWHAZ function in neural development

  • CRISPR/Cas9-engineered chicken YWHAZ mutant lines to study neurodevelopmental processes

  • Optogenetic approaches to manipulate YWHAZ-dependent signaling in real-time

  • Single-cell transcriptomics to map YWHAZ expression in developing neural circuits

• Immunological contexts:

  • Assessment of recombinant YWHAZ as an immunoprotective agent

  • Studies show recombinant YWHAZ provides protection in certain infection models

  • For example, r Ei-14-3-3 immunization achieved 86.13% oocyst reduction and 81.94% relative weight gain in a coccidiosis model

• Cross-disciplinary approaches:

  • Integration of in vivo models with computational approaches

  • Protein-protein interaction networks across developmental stages

  • Comparative immunological studies across species

  • Investigation of 14-3-3 proteins as potential vaccine components

• Novel methodologies:

  • CRISPR screens targeting YWHAZ regulatory elements

  • Spatial transcriptomics to map YWHAZ expression in tissues

  • Cryo-electron tomography to visualize YWHAZ-containing complexes in situ

These approaches are revealing new functions for YWHAZ beyond its classical roles in signaling and cell cycle regulation, with particular promise in neurological and immunological research areas .

How might understanding chicken YWHAZ inform therapeutic strategies for human diseases?

Chicken YWHAZ research has significant translational potential:

• Cancer therapeutics:

  • YWHAZ is frequently overexpressed in human cancers including bladder, gastric, and prostate cancer

  • Hazard ratios of 2.3 (95% CI: 1.003–5.304) have been reported in some cancers

  • Targeting strategies developed in chicken models could inform human cancer therapeutics

  • Specific inhibitors of YWHAZ-partner protein interactions could be developed based on conserved binding interfaces

• Neurodevelopmental disorders:

  • YWHAZ variants cause intellectual disability and global developmental delay in humans

  • YWHAZ deficiency in model organisms leads to neurodevelopmental defects

  • Insights from chicken models may help understand how YWHAZ dysfunction contributes to these disorders

• Infectious disease applications:

  • Studies show 14-3-3 proteins can provide cross-protective effects against different species of pathogens

  • Recombinant 14-3-3 proteins could be developed as adjuvants or immunomodulators

  • The high conservation (>99% amino acid sequence similarity) between related species suggests broad protective potential

• Target validation approaches:

  • Use of chicken models for initial validation of YWHAZ as a therapeutic target

  • Development of small molecule modulators of YWHAZ function based on structural insights

  • Investigation of YWHAZ in drug resistance mechanisms