Recombinant Chicken Protein AATF (AATF), partial

Basic Research Questions

• What is chicken AATF and what challenges exist in its identification?

  AATF (Apoptosis Antagonizing Transcription Factor) is a protein involved in transcriptional regulation, cell cycle control, DNA damage responses, and cell death pathways. The chicken AATF was initially considered absent in avian genomes. The identification challenges stemmed from its highly GC-rich sequence, which caused technical difficulties in PCR amplification and underrepresentation in next-generation sequencing data . The breakthrough in identification came through advanced data mining technologies and comparative genomic approaches, similar to the identification of other "missing" genes like erythropoietin and leptin in the chicken genome .

  Methodological approach: For researchers attempting to identify similar challenging avian genes, the recommended approach involves:

  • Using multiple overlapping shorter fragments for PCR amplification

  • Implementing specialized PCR protocols for GC-rich sequences

  • Employing comparative genomics with other avian species and reptiles

  • Verifying sequence through RNA isolation and RT-PCR from relevant tissues

• How does chicken AATF compare structurally and functionally to mammalian AATF?

  Chicken AATF shares approximately 45% homology with its mammalian counterpart in the extracellular region containing the TNF superfamily motif . The chicken AATF protein consists of 285 amino acids and contains an extended intracellular domain compared to its mammalian homologs, while maintaining a relatively well-conserved extracellular domain .

  Functionally, like mammalian AATF, chicken AATF is involved in:

  • Transcriptional regulation

  • Cell cycle control

  • DNA damage responses

  • Execution of cell death programs

  Methodological comparison: When studying evolutionary conservation, researchers should employ:

  • Multiple sequence alignment tools focusing on conserved domains

  • Phylogenetic analysis across species

  • Homology modeling of protein structure

  • Cross-species functional assays to determine conserved activities

• What expression systems are effective for producing recombinant chicken AATF?

  Due to the highly GC-rich nature of chicken AATF, expression requires specialized approaches. Successful expression has been achieved using:

  • E. coli expression systems: Using codon-optimized sequences to avoid GC-rich regions, as demonstrated in search result where synthetic chTNF-α was expressed in HEK 293 cells after codon optimization .

  • Mammalian cell expression: HEK293 cells have been used successfully for expression following codon optimization .

  Methodological protocol:

  1. Perform codon optimization to reduce GC content while maintaining amino acid sequence

  2. Clone into expression vectors with strong promoters (e.g., pcDNA3.1)

  3. Transform/transfect host cells and verify expression using Western blot

  4. For E. coli systems, consider fusion tags (GST, His) to enhance solubility

  5. Confirm protein identity using mass spectrometry (as performed in )

• What bioassays can be used to verify functional activity of recombinant chicken AATF?

  Functional verification of recombinant chicken AATF can be performed using:

  1. Reporter cell assays: As demonstrated in search result , recombinant chicken AATF activated a quail CEC-NFκB-luciferase reporter cell line with similar potency as recombinant chIL-1 .

  2. Thermal stability testing: Heat treatment (80°C for 5 min) to distinguish between biological activity and potential contaminants like LPS .

  3. Cell proliferation assays: Measuring impact on cellular proliferation, as AATF knockdown has been shown to decrease cell proliferation by 41% in mammalian models .

  Methodological considerations:

  • Include positive controls (e.g., known cytokine activators)

  • Perform dose-response curves to establish potency

  • Include heat-inactivated samples as negative controls

  • Verify specificity using neutralizing antibodies

• How can chicken cell models be established for studying AATF function?

  Researchers can establish chicken cell models through:

  1. Primary cell cultures: Isolation of monocyte-derived macrophages from chicken, which have been shown to express AATF upon LPS stimulation .

  2. Immortalized cell lines: Generation of immortalized chicken cell lines using chicken telomerase reverse transcriptase (chTERT) and chicken telomerase RNA (chTR) as demonstrated in search result , which showed successful immortalization of chicken preadipocytes .

  3. Established chicken cell lines: Using available chicken cell lines such as DF-1 fibroblasts or MSB-1 lymphoid cells for transfection studies .

  Methodological approach for immortalization:

  1. Isolate primary chicken cells of interest

  2. Perform retroviral transduction of chTERT and/or chTR

  3. Select transduced cells and verify telomerase activity

  4. Confirm phenotypic stability through multiple passages

  5. Validate that cells maintain relevant AATF response pathways

Advanced Research Questions

• What experimental design strategies are effective for studying chicken AATF using fractional factorial designs?

  When researching complex protein interactions like those involving chicken AATF, fractional factorial designs offer efficient experimental approaches:

  Methodological guidance:

  • Use 2^(k-p) fractional factorial design notation where:

    • l = number of levels per treatment factor (typically 2 for AATF experiments)

    • k = number of treatment factors (e.g., concentration, temperature, cell type, cofactors)

    • p = number of interactions that are confounded

  • For a 4-factor AATF experiment with 1 confounded factor:

    • Design classification: 2^(4-1) fractional factorial design

    • Required trials: 1/(2^1) or 1/2 of full factorial (8 instead of 16 trials)

  Implementation strategy:

  1. Select key factors affecting AATF activity (e.g., temperature, pH, cell type, stimulation)

  2. Determine high/low levels for each factor

  3. Create alias structure to identify which interactions will be confounded

  4. Generate design matrix using statistical software

  5. Perform experiments following the matrix

  6. Analyze main effects first, then investigate potential interactions

  This approach is particularly valuable when studying complex signaling pathways involving AATF where multiple factors may interact.

• How can CRISPR/Cas9 gene editing be optimized for chicken AATF functional studies?

  CRISPR/Cas9 gene editing offers powerful approaches for studying chicken AATF function:

  Methodological protocol based on search results:

  1. Design of guide RNAs (gRNAs):

     • Target non-GC-rich regions of the AATF gene to improve efficiency

     • Use chicken genome databases to ensure specificity

     • Design multiple gRNAs targeting different exons

  2. Delivery systems for chicken cells:

     • For DF-1 cells: nucleofection or lipofection protocols

     • For primordial germ cells (PGCs): nucleofection followed by antibiotic selection

  3. Verification of editing:

     • PCR amplification of targeted regions followed by sequencing

     • Western blot analysis of AATF protein expression

     • RT-PCR for mRNA expression analysis

  4. Phenotypic analysis:

     • Cell proliferation assays

     • Apoptosis assays

     • Gene expression analysis of downstream targets

  Case example: Research has successfully used CRISPR/Cas9 to generate chicken DF-1 cells lacking ANP32B while retaining ANP32A expression, demonstrating the feasibility of targeted gene editing in chicken cell lines .

• What are the optimal approaches for analyzing chicken AATF interactions with the DNA damage response pathway?

  AATF has been implicated in DNA damage response pathways. For chicken AATF studies:

  Experimental approach:

  1. Induction of DNA damage:

     • Use ionizing radiation, as demonstrated in MSB-1 cells

     • Chemical DNA-damaging agents (e.g., etoposide, doxorubicin)

  2. Analysis of AATF phosphorylation:

     • Immunoprecipitation followed by Western blot with phospho-specific antibodies

     • Mass spectrometry to identify phosphorylation sites

     • In vitro kinase assays with purified checkpoint kinases (Chk1/2)

  3. Subcellular localization studies:

     • Immunofluorescence microscopy to track AATF translocation

     • Cell fractionation followed by Western blot

  4. Functional relationship analysis:

     • Co-immunoprecipitation to identify interaction partners

     • ChIP-seq to identify DNA binding sites of AATF

     • RNA-seq to analyze transcriptional changes

  Research insight: Studies with the chicken anemia virus (CAV) protein apoptin revealed that DNA damage signaling through the ATM-Chk2 pathway induces protein translocation from cytoplasm to nucleus, where it induces apoptosis . This mechanism may be relevant for understanding chicken AATF function in DNA damage response.

• How can recombinant viral vectors be optimized for chicken AATF expression or knockdown studies?

  Viral vectors provide efficient tools for studying chicken AATF:

  Methodological approaches:

  1. Retroviral systems:

     • Demonstrated success in chicken cell transduction

     • Protocol: Clone optimized AATF (or shRNA) sequence into retroviral vector, produce virus in packaging cells, transduce target chicken cells, select stable integrants

  2. Avian paramyxovirus vectors:

     • APMV-3 has been successfully used as a vector in chicken cells

     • Advantages: Replicates well in chickens and can express foreign proteins

     • Protocol for recombinant APMV-3 generation:

       1. Clone AATF into APMV-3 vector using reverse genetics system

       2. Co-transfect with support plasmids into HEp-2 cells

       3. Recover virus from transfected cells

       4. Amplify in embryonated chicken eggs

       5. Verify by sequencing

  3. Newcastle Disease Virus (NDV) vectors:

     • Successfully used to express foreign proteins in chicken cells

     • Similar protocol to APMV-3 but using NDV backbone

  Comparative data on vector efficiency:

  Vector System	Transduction Efficiency	Expression Duration	In vivo Applicability	Reference
  Retroviral	60-80% in dividing cells	Stable/permanent	Limited
  APMV-3	High in respiratory/systemic	1-2 weeks	Excellent
  NDV	High in respiratory tract	1-2 weeks	Good

• What are the contradictions in AATF function across different cell types and how can these be addressed experimentally?

  Research has revealed seemingly contradictory roles for AATF across different cell types:

  Contradictory findings:

  • In cancer cells: AATF acts as an anti-apoptotic factor, with knockdown inducing apoptosis (30% in MCF-7 breast cancer cells)

  • In normal cells: AATF knockdown produced only 18% apoptosis induction in non-malignant MCF-10A cells

  • In viral systems: AATF/similar proteins can have pro-apoptotic functions in specific contexts

  Experimental design to address contradictions:

  1. Comparative protein interaction studies:

     • Perform IP-MS in multiple cell types to identify differential binding partners

     • Compare post-translational modifications across cell types

  2. Domain-specific functional analysis:

     • Generate truncated versions of chicken AATF to identify functional domains

     • Use domain-swapping experiments between chicken and mammalian AATF

  3. Context-dependent transcriptional profiling:

     • RNA-seq analysis of different cell types expressing or lacking AATF

     • ChIP-seq to identify differential DNA binding patterns

  4. Cell-type specific knockdown/knockout:

     • Use tissue-specific promoters to drive shRNA expression

     • Conditional knockout systems (e.g., Cre-loxP) in animal models

  Proposed experimental framework:

  1. Establish identical knockdown efficiency across multiple cell types

  2. Perform parallel omics analyses (proteomics, transcriptomics)

  3. Identify differential interaction networks

  4. Validate key differential interactions using co-IP and functional assays

• How can interspecies differences in AATF function be systematically analyzed through comparative studies?

  Interspecies variation in AATF function provides valuable insights:

  Methodological approach:

  1. Cross-species sequence and structure analysis:

     • Multiple sequence alignment of AATF from birds, mammals, reptiles

     • Identification of conserved domains versus variable regions

     • Homology modeling to predict structural differences

  2. Complementation assays:

     • Express chicken AATF in mammalian AATF-knockout cells

     • Express mammalian AATF in chicken AATF-knockout cells

     • Measure rescue of phenotypes (cell cycle, apoptosis, transcription)

  3. Chimeric protein analysis:

     • Generate chimeric proteins containing domains from chicken and mammalian AATF

     • Identify which domains confer species-specific functions

  4. Comparative interactome analysis:

     • BioID or proximity labeling coupled with mass spectrometry

     • Compare interaction partners between chicken and mammalian AATF

     • Identify conserved versus species-specific interactions

  Research insight: The study of ANP32 proteins in chicken revealed species-specific differences in supporting influenza virus polymerase activity, with chicken ANP32A but not ANP32B supporting activity, unlike in mammalian systems . This provides a model for studying species-specific protein functions.

• What are the optimal experimental designs for studying partial versus full-length chicken AATF functions?

  Understanding the functional differences between partial and full-length chicken AATF requires specific experimental approaches:

  Methodological strategy:

  1. Domain mapping and construction:

     • Analyze protein domains using bioinformatics

     • Generate expression constructs for:

       • Full-length AATF

       • N-terminal fragment (partial)

       • C-terminal fragment

       • Internal domain deletions

  2. Functional comparison assays:

     • Subcellular localization using fluorescent tags

     • Protein-protein interaction analysis via co-IP

     • Transcriptional activity using reporter assays

     • Cell cycle and apoptosis effects

  3. Structural biology approaches:

     • Circular dichroism to assess secondary structure

     • Limited proteolysis to identify domain boundaries

     • X-ray crystallography or Cryo-EM for structure determination

  Example experimental design:

  Construct	Expected Size	Domains Present	Assays
  Full-length	285 aa	All domains	All functional assays
  N-terminal (aa 1-150)	150 aa	Nuclear localization	Localization, DNA binding
  C-terminal (aa 151-285)	135 aa	Protein interaction	Co-IP, apoptosis assays
  Internal deletion	Variable	Domain-specific	Function-specific assays

  This approach allows systematic characterization of domain-specific functions and comparison between partial and complete protein activities.

• How can mass spectrometry methods be optimized for chicken AATF identification and characterization?

  Mass spectrometry provides powerful tools for AATF analysis:

  Methodological protocol:

  1. Sample preparation optimization:

     • For recombinant AATF: In-gel digestion following SDS-PAGE

     • For endogenous AATF: Immunoprecipitation followed by on-bead digestion

     • For complex samples: Fractionation methods (SCX, high-pH RP)

  2. MS analysis strategies:

     • Use multiple proteases (trypsin, chymotrypsin, Glu-C) to increase coverage

     • Apply specialized methods for GC-rich proteins

     • For post-translational modifications: Phospho-enrichment using TiO2 or IMAC

  3. Data analysis approach:

     • Search against chicken-specific databases

     • Consider sequence variants and isoforms

     • Use de novo sequencing for novel peptides

  4. Verification methods:

     • Parallel reaction monitoring (PRM) for targeted quantification

     • Heavy-labeled peptide standards for absolute quantification

  Case study: In search result , mass spectrometry successfully identified three unique peptides from recombinant chicken AATF protein with sequence coverage of 10.2%, a MaxQuant Score of 83.5, and a Q-value (false-positive probability) of zero .