Recombinant Chicken Polyadenylate-binding protein-interacting protein 2 (PAIP2)

What is PAIP2 and what is its primary function in cellular processes?

PAIP2 (Polyadenylate-binding protein-interacting protein 2) is a regulatory protein that primarily functions as an inhibitor of PABP1 (Poly(A) binding protein 1). It plays a crucial role in translation regulation by preventing PABP1 from binding to poly(A) RNA and destabilizing the circularized mRNA structure . This inhibitory activity makes PAIP2 an important negative regulator of protein synthesis. In physiological contexts, PAIP2 contributes to various biological processes including synaptic plasticity regulation, memory formation, spermatogenesis, and innate defense against viral infections by restricting viral protein synthesis . PAIP2 achieves these regulatory functions through specific protein-protein interactions with PABP1, which in turn affects mRNA stability and translation efficiency.

How does chicken PAIP2 differ structurally and functionally from mammalian PAIP2?

While the search results don't explicitly compare chicken PAIP2 to mammalian orthologs, research shows that PAIP2 contains highly conserved interaction domains across species. All PAIP2 proteins contain two key interaction motifs: PAM1 and PAM2 . The PAM1 domain binds to the RNA recognition motifs (RRMs) of PABP1 and is characterized by numerous negatively charged residues that alter PABP1 conformation to prevent poly(A) RNA binding . The PAM2 motif interacts with the MLLE domain of PABP1, with a conserved phenylalanine residue that is critical for this interaction .

Any species-specific differences would likely be found in non-conserved regions, potentially affecting binding affinities or regulatory mechanisms specific to avian translation systems. Researchers working with chicken PAIP2 should consider these potential differences when extrapolating findings across species.

What are the optimal expression systems for producing recombinant chicken PAIP2?

Based on current research practices with similar proteins, several expression systems can be considered for recombinant chicken PAIP2 production:

E. coli Expression System:

• Advantages: High yield, cost-effective, rapid production

• Methodology: Clone the chicken PAIP2 cDNA into a bacterial expression vector containing an N-terminal affinity tag (His, GST, etc.)

• Considerations: May require optimization of codon usage for bacterial expression, and the protein might lack post-translational modifications

Insect Cell Expression System:

• Advantages: Provides eukaryotic post-translational modifications

• Methodology: Baculovirus-mediated expression in Sf9 or High Five insect cells

• Considerations: Offers a compromise between bacterial systems (yield) and mammalian systems (proper folding)

Mammalian Cell Expression:

• Advantages: Most likely to produce properly folded protein with all necessary modifications

• Methodology: Transient or stable transfection in HEK293 or CHO cells

• Considerations: Lower yield but potentially higher biological activity

The optimal system would depend on the specific research questions and whether post-translational modifications are critical for the intended application.

What purification strategies yield the highest purity and activity for recombinant chicken PAIP2?

A multi-step purification strategy is recommended for obtaining high-purity active recombinant chicken PAIP2:

• Initial Capture: Affinity chromatography using the appropriate resin based on the fusion tag:

  • His-tagged PAIP2: Ni-NTA or IMAC chromatography

  • GST-tagged PAIP2: Glutathione Sepharose chromatography as indicated in search results

• Intermediate Purification: Ion exchange chromatography

  • Given PAIP2's negative charge properties mentioned in the PAM1 domain , anion exchange chromatography at appropriate pH would be suitable

• Polishing Step: Size exclusion chromatography

  • To remove aggregates and ensure homogeneity

• Tag Removal: If necessary, cleave the affinity tag using a specific protease

  • Verify tag removal by SDS-PAGE and Western blot

• Activity Verification:

  • PABP1 binding assay using pull-down experiments similar to those described in search result

  • RNA competition assays to verify functional inhibition of PABP1-poly(A) interaction

Maintaining protein stability throughout purification is critical, so including appropriate buffers with stabilizing agents (glycerol, reducing agents) is recommended.

How can researchers quantitatively measure the binding affinity between recombinant chicken PAIP2 and PABP1?

Several quantitative methods can be employed to measure the binding affinity between recombinant chicken PAIP2 and PABP1:

Surface Plasmon Resonance (SPR):

• Methodology: Immobilize purified PABP1 on a sensor chip and measure real-time binding kinetics of PAIP2 at various concentrations

• Output: Association (kon) and dissociation (koff) rate constants and equilibrium dissociation constant (KD)

• Advantage: Provides both kinetic and thermodynamic parameters

Isothermal Titration Calorimetry (ITC):

• Methodology: Directly measure heat changes during binding to determine thermodynamic parameters

• Output: Binding stoichiometry, enthalpy change (ΔH), and KD

• Advantage: No immobilization or labeling required

Microscale Thermophoresis (MST):

• Methodology: Measure changes in thermophoretic mobility upon binding

• Output: KD values in solution

• Advantage: Requires small sample amounts

Co-Immunoprecipitation with Quantification:

• Methodology: Similar to methods described in search result , using anti-GFP antibody to immunoprecipitate Paip2-GFP and detect associated PABP1

• Output: Semi-quantitative binding data

• Advantage: Closer to physiological conditions

These methods would allow researchers to compare chicken PAIP2's binding properties with those of other species and mutant versions.

What are the critical residues in chicken PAIP2 responsible for PABP1 interaction?

Based on the search results, PAIP2 interacts with PABP1 through two distinct motifs, and these interactions involve specific critical residues:

PAM1 Domain Interaction with RRM domains of PABP1:

• Characterized by numerous negatively charged residues (Asp, Glu)

• These residues change the conformation of PABP1 and exclude poly(A) RNA binding

• Critical for the inhibitory function of PAIP2

PAM2 Domain Interaction with MLLE domain of PABP1:

• A phenylalanine residue is critical for PAM2/MLLE interaction

• This interaction is highly conserved across species

To identify the exact critical residues in chicken PAIP2 specifically, researchers should consider:

• Sequence alignment of chicken PAIP2 with well-characterized PAIP2 from other species

• Site-directed mutagenesis of conserved residues, particularly:

  • The key phenylalanine in the PAM2 motif

  • Negatively charged clusters in the PAM1 region

• Testing mutants for PABP1 binding using the methods described in 3.1

• Functional assays to determine the effect of mutations on translation inhibition

The resulting data would help create a comprehensive map of the chicken PAIP2-PABP1 interaction interface.

How does chicken PAIP2 specifically regulate translation initiation and termination?

Chicken PAIP2, like its counterparts in other species, plays a multifaceted role in translation regulation through its interaction with PABP1:

Translation Initiation Regulation:

• PAIP2 inhibits translation by preventing PABP1 from binding to poly(A) RNA and destabilizing the circularized mRNA structure

• This disrupts the interaction between PABP1 and eIF4G, weakening the mRNA closed-loop formation that enhances translation initiation

• The conformational change induced in PABP1 by PAIP2 binding through its PAM1 domain specifically excludes poly(A) RNA binding

Translation Termination Effects:

• PAIP2 prevents translation termination at premature termination codons by controlling PABP activity

• PAIP2 inhibits the activity of free PABP on translation termination in vitro

• It serves as an important regulator of readthrough at premature termination codons

These mechanisms allow PAIP2 to function as a negative regulator of translation under various physiological conditions, providing a fine-tuning mechanism for protein synthesis.

What is the role of chicken PAIP2 in viral defense mechanisms?

PAIP2 plays a significant role in innate defense against viral infections by restricting viral protein synthesis. This occurs through several mechanisms:

• Counteracting Virus-Induced PABP1 Increases: PAIP2 serves as an innate defense to restrict viral protein synthesis to counter virus-induced increases in PABP1

• Viral Countermeasures: Some viruses have evolved strategies to overcome this defense:

  • Certain viruses, like MDV1 (Marek's Disease Virus 1), produce microRNAs that target and repress PAIP2

  • These viral miRNAs downregulate PAIP2 expression through binding to multiple microRNA response elements (MREs) in the PAIP2 mRNA 3'UTR

  • The repression of PAIP2 indirectly contributes to increased levels of available active PABP1

• Impact on IRES-Mediated Translation: PAIP2 suppression can affect viral Internal Ribosome Entry Site (IRES) activity:

  • When viral miRNAs downregulate PAIP2, more active PABP1 becomes available

  • PABP1 can then interact with internal poly(A) sequences in viral IRES elements, enhancing viral translation

This interplay between PAIP2 and viral mechanisms represents an evolutionary arms race between host defense and viral countermeasures, making PAIP2 an important factor in understanding viral pathogenesis and potential antiviral strategies.

How can recombinant chicken PAIP2 be used to study translation dynamics in cell-free systems?

Recombinant chicken PAIP2 can serve as a valuable tool for studying translation dynamics in cell-free systems through the following methodologies:

In Vitro Translation Modulation Studies:

• Adding purified recombinant PAIP2 at different concentrations to rabbit reticulocyte lysate (RRL) or wheat germ extract containing reporter mRNAs

• Measuring dose-dependent inhibition of translation to establish structure-function relationships

• Comparing wild-type PAIP2 with mutant versions to identify key functional domains

Reconstituted Translation Systems:

• Using PAIP2 in conjunction with purified translation factors to reconstitute minimal translation systems

• Systematically analyzing the effect of PAIP2 on individual steps of translation (initiation, elongation, termination)

• Establishing the minimal components required for PAIP2-mediated regulation

Translation Termination and Readthrough Assays:

• Utilizing PAIP2 to study premature termination codon readthrough mechanisms as indicated in search result

• Measuring the effects of PAIP2 on translation termination efficiency at normal and premature stop codons

• Investigating the competition between PAIP2 and eRF3 for PABP binding and its impact on termination

PABP1 Conformational Studies:

• Using PAIP2 as a tool to induce specific conformational changes in PABP1

• Analyzing these structural changes using techniques like FRET or hydrogen-deuterium exchange mass spectrometry

• Correlating structural changes with functional outcomes in translation

These applications would provide valuable insights into the mechanisms of translation regulation that could potentially be extrapolated to in vivo systems.

What are the challenges in designing PAIP2 mutants for structure-function studies?

Designing PAIP2 mutants for structure-function studies presents several challenges that researchers should consider:

Structural Challenges:

• Limited Structural Information: Complete structural data for chicken PAIP2 is limited, making rational design of mutants challenging

• Multiple Interaction Domains: PAIP2 contains two distinct interaction domains (PAM1 and PAM2) , requiring careful consideration of how mutations in one domain might affect the other

• Conformational Flexibility: PAIP2 likely has regions of conformational flexibility that are difficult to predict and model

Functional Challenges:

• Dual Binding Interfaces: Mutations must be designed to distinguish between effects on PABP1 binding via RRM domains (PAM1) and MLLE domain (PAM2)

• Allosteric Effects: Mutations may cause unpredicted allosteric effects that alter protein function beyond the immediate binding site

• Separating Functions: Creating mutants that specifically affect one function (e.g., translation initiation) without affecting others (e.g., translation termination) is difficult

Technical Challenges:

• Protein Stability: Mutations may affect protein stability and solubility, particularly in the negatively charged PAM1 region

• Expression Systems: Different mutations may require optimization of expression conditions

• Assay Sensitivity: Developing sensitive assays to detect subtle functional changes resulting from mutations

Recommended Approach:

• Begin with alanine-scanning mutagenesis of conserved residues, particularly the critical phenylalanine in PAM2

• Create domain deletion mutants to assess the contribution of each domain

• Design mutations based on sequence conservation across species

• Employ multiple complementary assays to comprehensively assess mutant function

This methodical approach would help overcome these challenges and provide valuable insights into PAIP2 structure-function relationships.

How does PAIP2 expression or activity correlate with disease states in avian models?

While the search results don't provide specific information about PAIP2 in avian disease models, its role in fundamental cellular processes suggests potential involvement in various pathological conditions:

Viral Infections:

• PAIP2 serves as an innate defense mechanism against viral infections by restricting viral protein synthesis

• Some viruses, like Marek's Disease Virus 1 (MDV1), have evolved countermeasures, producing microRNAs that target and repress PAIP2

• The virus-induced suppression of PAIP2 contributes to enhanced viral IRES activity and increased viral replication

• This suggests that PAIP2 dysregulation may correlate with susceptibility to or severity of viral infections in avian models

Cancer Models:

• Since PAIP2 regulates translation, which is often dysregulated in cancer, altered PAIP2 expression might play a role in avian tumor models

• The interaction between PAIP2 and PABP1 affects global protein synthesis, potentially influencing cell proliferation and oncogenic transformation

Neurological Disorders:

• PAIP2 contributes to the control of synaptic plasticity and memory formation

• Dysregulation might be associated with cognitive or neurological disorders in avian models

Researchers investigating these correlations should consider:

• Analyzing PAIP2 expression levels in healthy versus diseased tissues

• Examining the effect of PAIP2 knockdown or overexpression on disease progression

• Investigating potential post-translational modifications of PAIP2 in disease states

Can recombinant PAIP2 be utilized to develop antiviral strategies against avian pathogens?

The role of PAIP2 in viral defense mechanisms suggests potential applications in developing antiviral strategies:

Potential Antiviral Approaches:

• PAIP2 as a Therapeutic Protein:

  • Delivery of recombinant PAIP2 or PAIP2-derived peptides could enhance cellular defense against viruses that target PABP1

  • Specifically designed PAIP2 variants with enhanced stability or binding properties could serve as potent inhibitors of viral translation

• Targeting Viral miRNA-PAIP2 Interactions:

  • The identification of viral miRNAs that target PAIP2, such as those from MDV1 , provides potential targets for intervention

  • Antisense oligonucleotides or miRNA sponges designed to neutralize these viral miRNAs could prevent PAIP2 downregulation

  • This approach would preserve PAIP2's natural antiviral activity

• Small Molecule Modulators:

  • Developing small molecules that mimic PAIP2's interaction with PABP1 could provide pharmacological tools to restrict viral protein synthesis

  • Alternatively, compounds that protect PAIP2 from virus-induced degradation or repression could enhance antiviral responses

Challenges and Considerations:

• Delivery Methods: Developing effective delivery systems for recombinant PAIP2 or PAIP2-modulating compounds

• Specificity: Ensuring that interventions specifically affect viral translation without disrupting normal cellular protein synthesis

• Resistance Development: Viruses might evolve resistance to PAIP2-based therapies through mutations in their RNA elements or miRNAs

Research Approach:

• Screen for compounds that stabilize PAIP2-PABP1 interactions in the presence of viral factors

• Test recombinant PAIP2 variants for enhanced antiviral activity in cell culture models

• Develop targeted delivery systems for PAIP2 or PAIP2-modulating compounds to affected tissues

This research direction could lead to novel antiviral strategies particularly valuable for economically important avian diseases.

What are the best methods for detecting endogenous versus recombinant PAIP2 in experimental systems?

Distinguishing between endogenous and recombinant PAIP2 is crucial for accurate experimental interpretation. Several complementary methods can be employed:

Immunological Methods:

• Western Blotting:

  • For recombinant tagged PAIP2: Use antibodies against the tag (His, GST, etc.)

  • For distinguishing endogenous vs. recombinant: Use antibodies that recognize species-specific epitopes if using chicken PAIP2 in non-avian cells

  • Quantification: Densitometry analysis with appropriate standards

• Immunofluorescence:

  • Localization studies using tag-specific antibodies for recombinant protein

  • Co-localization studies with PABP1 to assess functional interaction

  • Super-resolution microscopy for detailed subcellular localization

Mass Spectrometry-Based Approaches:

• Targeted Proteomics:

  • Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) assays

  • Designed to detect peptides unique to recombinant PAIP2 (e.g., junction peptides with tags)

  • Absolute quantification using isotopically labeled standards

• Post-Translational Modification Analysis:

  • Identifying differences in modification patterns between endogenous and recombinant proteins

  • Particularly useful if the recombinant protein lacks specific modifications

Functional Assays:

• RNA-Protein Binding Assays:

  • UV crosslinking studies to assess interactions with specific RNA targets

  • Electrophoretic mobility shift assays (EMSA) to detect protein-RNA complexes

• PABP1 Interaction Assays:

  • Co-immunoprecipitation experiments as mentioned in search result

  • Pull-down assays using tagged recombinant proteins

These methods, particularly when used in combination, provide comprehensive detection and distinction between endogenous and recombinant PAIP2 proteins in experimental systems.

What are the critical controls needed when studying PAIP2-PABP1 interactions in different experimental systems?

When studying PAIP2-PABP1 interactions, several critical controls are necessary to ensure experimental validity and interpretability:

Protein-Specific Controls:

• Expression Level Controls:

  • Titration experiments to assess the effect of varying PAIP2:PABP1 ratios

  • Western blot quantification to ensure physiologically relevant expression levels

• Mutant Controls:

  • PAIP2 mutants lacking functional PAM1 or PAM2 domains

  • PABP1 mutants with disrupted RRM or MLLE domains

  • These controls help establish specificity of observed interactions

• Competition Controls:

  • Including known competitors like eRF3 which competes with PAIP2 for PABP1 binding

  • Demonstrating specificity through competitive binding assays

Experimental System Controls:

• In Vitro Binding Assays:

  • BSA or unrelated proteins as negative controls

  • Known PABP1 interactors as positive controls

  • Varying buffer conditions to assess interaction stability

• Cell-Based Assays:

  • Cell lines with PAIP2 or PABP1 knockdown/knockout

  • Rescue experiments with wild-type versus mutant proteins

  • Controls for cell type-specific effects

• RNA-Dependent Interaction Controls:

  • RNase treatment to determine RNA-dependence of interactions

  • Poly(A) RNA competition assays to assess functional consequences

  • Comparing free PABP1 versus poly(A)-bound PABP1 interactions with PAIP2

Technical Controls:

• Antibody Controls:

  • Isotype controls for immunoprecipitation experiments

  • Pre-immune serum controls

  • Validation of antibody specificity with recombinant proteins

• Tag Interference Controls:

  • Comparing N-terminal versus C-terminal tagged versions

  • Tag-only controls to rule out tag-mediated interactions

  • Comparing tagged and untagged proteins when possible

Implementing these controls ensures robust and interpretable results when studying the complex interactions between PAIP2 and PABP1 across different experimental systems.

What are the most pressing unanswered questions in chicken PAIP2 research?

Despite the advances in understanding PAIP2 function, several critical questions remain unanswered, particularly regarding chicken PAIP2:

• Species-Specific Mechanisms: How do the functional mechanisms of chicken PAIP2 differ from mammalian counterparts, particularly in translation regulation contexts specific to avian systems?

• Regulation of PAIP2: What are the upstream regulators of chicken PAIP2 expression and activity, and how do these change under different physiological conditions or stresses?

• Post-Translational Modifications: What PTMs affect chicken PAIP2 function, and how do these modifications regulate its interaction with PABP1 and other potential binding partners?

• Tissue-Specific Functions: Does chicken PAIP2 have tissue-specific roles in avian systems, particularly in immune cells, neurons, or reproductive tissues where PAIP2 has demonstrated important functions in mammals ?

• Role in Development: What is the developmental expression pattern and function of PAIP2 in avian embryogenesis?

• Viral Interactions Beyond MDV1: While MDV1 miRNAs target PAIP2 , do other avian viruses interact with PAIP2, and what are the mechanisms involved?

• Additional Binding Partners: Beyond PABP1, what other proteins interact with chicken PAIP2, and what are the functional consequences of these interactions?

• Therapeutic Potential: Can PAIP2 modulation be developed as a therapeutic strategy for avian diseases, particularly viral infections?

Addressing these questions will require interdisciplinary approaches combining structural biology, cell biology, virology, and systems biology to fully understand the complex roles of PAIP2 in avian systems.

What emerging technologies might advance research on recombinant chicken PAIP2?

Several emerging technologies show promise for advancing research on recombinant chicken PAIP2:

• CRISPR/Cas9 Genome Editing:

  • Generation of PAIP2 knockout or knock-in chicken cell lines

  • Introduction of tagged endogenous PAIP2 for live-cell imaging

  • Creation of domain-specific mutations to assess function in native context

• Cryo-Electron Microscopy (Cryo-EM):

  • Structural determination of PAIP2-PABP1 complexes at near-atomic resolution

  • Visualization of PAIP2 interactions with translation machinery

  • Analysis of conformational changes induced by PAIP2 binding to PABP1

• Proximity Labeling Proteomics:

  • BioID or APEX2 fusion proteins to identify novel PAIP2 interactors in avian cells

  • Spatial and temporal mapping of PAIP2 interaction networks

  • Comparison between healthy and stressed/infected conditions

• Single-Molecule Techniques:

  • FRET or single-molecule pull-down assays to study PAIP2-PABP1 interaction dynamics

  • Optical tweezers to measure binding/unbinding forces

  • Single-molecule translation assays to directly observe PAIP2 effects

• RNA-Protein Interaction Mapping:

  • CLIP-seq (Crosslinking immunoprecipitation-sequencing) to identify RNAs associated with PAIP2-PABP1 complexes

  • RNA maps of translation efficiency in the presence/absence of PAIP2

  • Structure probing of mRNAs affected by PAIP2 activity

• Microfluidics and Organ-on-Chip:

  • High-throughput screening of PAIP2 variants or modulators

  • Complex tissue models to study PAIP2 function in tissue-specific contexts

  • Real-time analysis of PAIP2 activity under varying conditions

• AI and Computational Approaches:

  • Machine learning prediction of PAIP2 interaction sites and functional domains

  • Molecular dynamics simulations of PAIP2-PABP1 interactions

  • Systems biology modeling of translation regulation networks involving PAIP2

These emerging technologies, particularly when used in combination, have the potential to significantly advance our understanding of chicken PAIP2 structure, function, and regulation, opening new avenues for both basic research and applied biotechnology.

What are the recommended expression constructs and conditions for optimal recombinant chicken PAIP2 production?

Based on available research and protein expression principles, the following recommendations are provided for optimal recombinant chicken PAIP2 production:

Expression Constructs Design:

Expression Conditions for E. coli:

Insect Cell Expression:

These recommendations provide a starting point for establishing an optimal expression system for recombinant chicken PAIP2, which may require further optimization based on specific research needs.

What are the key considerations for designing in vitro experiments to study PAIP2-mediated translation regulation?

When designing in vitro experiments to study PAIP2-mediated translation regulation, researchers should consider several key factors:

Translation System Design:

• Cell-Free Translation Systems:

  • Rabbit reticulocyte lysate (RRL): Mammalian system with complete translation machinery

  • Wheat germ extract: Plant-based alternative with lower background

  • Nuclease-treated lysates: For cap-dependent translation studies

  • Consider using homologous avian systems when studying chicken PAIP2

• Reporter mRNAs:

  • Include both capped and uncapped reporter mRNAs

  • Compare poly(A)+ and poly(A)- mRNAs to assess poly(A)-dependent effects

  • Include mRNAs with premature termination codons to study termination effects

  • Use bicistronic reporters to study effects on IRES-dependent translation

PAIP2 Concentration Effects:

• Titration Experiments:

  • Use a range of PAIP2 concentrations (1 nM to 1 μM)

  • Determine IC50 for translation inhibition

  • Compare wild-type PAIP2 with domain mutants

• Competition Studies:

  • Include eRF3 at various concentrations to study competition for PABP binding

  • Test PABP1 rescue of PAIP2 inhibition

RNA-Protein Interaction Analysis:

• Binding Assays:

  • Electrophoretic mobility shift assays (EMSA)

  • Filter binding assays

  • SPR or BLI for real-time binding analysis

  • Consider RNA length and sequence context effects

• Functional Consequences:

  • Measure PABP1 binding to poly(A) in the presence/absence of PAIP2

  • Test if PAIP2 affects poly(A) protection from nucleases

Translation Readout Methods:

• Quantification Options:

  • Luciferase assays for sensitive detection

  • Radiolabeled amino acid incorporation for direct measurement

  • Western blotting for protein size verification

  • Ribosome profiling to assess ribosome positioning

• Kinetic Measurements:

  • Time-course experiments to distinguish effects on initiation vs. elongation

  • Pulse-chase studies to measure effects on protein synthesis rates

Experimental Controls:

• Positive Controls:

  • Known translation inhibitors (cycloheximide, puromycin)

  • Translation stimulators (PABP1 alone)

• Negative Controls:

  • Inactive PAIP2 mutants

  • Unrelated proteins of similar size/charge

• System Validation:

  • Cap analog competition to verify cap-dependent translation

  • Poly(A) competition to verify poly(A)-dependent effects

These considerations will help researchers design robust in vitro experiments to elucidate the mechanisms of PAIP2-mediated translation regulation, particularly in the context of avian systems and recombinant chicken PAIP2.