Recombinant Chicken Hsc70-interacting protein (ST13)

What is chicken Hsc70-interacting protein (ST13) and what is its primary function?

Chicken Hsc70-interacting protein (ST13) is an adaptor protein that mediates the association between heat shock proteins HSP70 and HSP90. It functions as a regulatory co-chaperone that modulates the activity of molecular chaperones, particularly Hsc70. ST13 plays a critical role in the assembly process of glucocorticoid receptor complexes, which requires multiple molecular chaperones working together . The protein stabilizes the ADP state of HSC70, which maintains a high affinity for substrate proteins, thereby contributing to protein quality control mechanisms . ST13 also has its own chaperone activity, which may contribute to the interaction of HSC70 with various target proteins.

How does chicken ST13 contribute to protein quality control in avian cells?

ST13 contributes to protein quality control through its regulatory effect on Hsc70 activity. Studies suggest that ST13 interacts with Hsc70-4 to mediate the degradation of misfolded or unfolded proteins . This mechanism is part of a larger protein quality control system that prevents the accumulation of potentially toxic protein aggregates. The ST13-Hsc70 complex recognition of substrate proteins appears to be driven by exposure of hydrophobic regions in misfolded proteins, allowing the chaperone system to either refold the proteins or target them for degradation if refolding fails. In other organisms, studies show that ST13 (HIP) binds the ATPase domains of at least two HSC70 molecules in a manner dependent on activation of HSC70 ATPase by HSP40, which indicates similar mechanisms likely exist in avian cells .

How does the structure of ST13 relate to its function in the chaperone system?

The functional architecture of ST13 is critical to its role as a co-chaperone. Based on available sequence data (e.g., the mouse ST13 AA 1-371 shown in search result ), ST13 contains:

• An N-terminal region involved in oligomerization

• Multiple tetratricopeptide repeat (TPR) domains that facilitate protein-protein interactions with Hsc70

• A central region that interacts with the ATPase domain of Hsc70

• A C-terminal region with charged residues

This structural organization allows ST13 to modulate Hsc70's ATPase activity and client binding. The TPR domains particularly contribute to the specificity of interactions with Hsc70, while the ability to form oligomers enables ST13 to simultaneously interact with multiple Hsc70 molecules, potentially enhancing the efficiency of chaperone activity .

How do different expression systems affect the post-translational modifications of recombinant chicken ST13?

Expression systems significantly impact the post-translational modifications (PTMs) of recombinant proteins, including chicken ST13. A comparative study of DnaK (bacterial Hsp70) expressed in different systems demonstrated:

These differences in PTMs significantly altered the protein's electrostatic landscape and functional properties . For recombinant chicken ST13, similar principles apply—eukaryotic expression systems like P. pastoris would introduce more extensive PTMs, particularly phosphorylation, compared to prokaryotic systems like E. coli. These modifications can dramatically alter ST13's binding affinity for Hsc70 and other interaction partners, potentially affecting experimental outcomes .

What purification strategies yield the highest purity and activity for recombinant chicken ST13?

The optimal purification strategy for recombinant chicken ST13 typically involves:

• Affinity chromatography: Using tags such as His, Strep, or GST for initial capture, achieving >80% purity

• Secondary purification: Ion-exchange chromatography based on ST13's charge properties

• Polishing step: Size-exclusion chromatography (SEC) to remove aggregates and achieve >90% purity

A successful approach reported for ST13 involved expression in HEK-293 cells with a His tag and purification using one-step affinity chromatography, yielding >90% purity as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC . Alternative tags such as Strep tag have also been used successfully for ST13 purification from cell-free protein synthesis systems, yielding 70-80% purity .

The choice of buffer composition is critical for maintaining ST13 activity, with considerations for pH (typically 7.0-8.0), salt concentration (usually 150-300 mM NaCl), and the inclusion of stabilizing agents such as glycerol (5-10%). Storage at -80°C helps maintain activity for up to 12 months .

How can recombinant chicken ST13 be used to study protein folding mechanisms?

Recombinant chicken ST13 serves as a valuable tool for investigating protein folding mechanisms through several methodological approaches:

• Reconstituted chaperone systems: Purified ST13 can be combined with Hsc70, Hsp40, and Hsp90 to study how this co-chaperone affects the folding efficiency of model substrate proteins. By measuring folding kinetics with and without ST13, researchers can determine its specific contribution to the chaperone pathway.

• ATPase activity assays: Since ST13 regulates Hsc70's ATPase activity, researchers can measure how varying concentrations of ST13 affect ATP hydrolysis rates and correlate this with folding outcomes.

• Client protein specificity analysis: ST13 may influence which clients Hsc70 preferentially binds. Techniques like the ubiquitin-activated interaction trap (UBAIT) fusion system can identify changes in client specificity when ST13 is present or absent .

• Structural biology approaches: Cryo-EM and X-ray crystallography of ST13-Hsc70 complexes, with and without client proteins, can reveal structural changes that occur during the folding process.

These approaches provide mechanistic insights into how ST13 contributes to protein folding pathways in avian systems, which may differ from mammalian systems in subtle but important ways.

What is the role of chicken ST13 in viral infection processes?

Research suggests that chicken ST13, through its interaction with Hsc70, may play a significant role in viral infection processes in avian species. Studies on Infectious Bursal Disease Virus (IBDV) provide insight into potential mechanisms:

• Viral replication complex formation: Chicken Hsp70 colocalizes with viral double-stranded RNA (dsRNA) and directly interacts with viral proteins VP2 and VP3, suggesting involvement in forming the replication and transcription complex . As a regulator of Hsc70/Hsp70, ST13 likely influences this process.

• Modulation of chaperone activity: ST13 stabilizes the ADP-bound state of Hsc70, which has high substrate affinity. This regulation could affect Hsc70's interaction with viral components during infection.

• Viral protein folding assistance: ST13-Hsc70 interactions may facilitate the proper folding of viral proteins, which is essential for virion assembly.

Experimental evidence shows that overexpression of cHsp70 promotes IBDV production, while knockdown reduces viral production . Given ST13's role in regulating Hsp70/Hsc70 activity, targeting ST13 represents a potential antiviral strategy by disrupting chaperone-dependent viral processes. Research utilizing recombinant ST13 in viral infection models could further elucidate these mechanisms and identify potential therapeutic targets.

What methodologies are most effective for assessing the interaction between recombinant chicken ST13 and Hsc70?

Several complementary methodologies provide robust assessment of ST13-Hsc70 interactions:

• Ubiquitin-mediated proximity tagging: The UBAIT system enables quantitative identification of transient interactions by covalently linking ST13 to Hsc70 through ubiquitin fusion proteins. This approach has successfully identified client proteins of HSP70 and HSC70 in human cells .

• Pull-down assays with purified components: Using purified recombinant ST13 (with affinity tags) and Hsc70 to directly assess binding stoichiometry, affinity constants, and the effects of nucleotides (ATP vs. ADP).

• Surface Plasmon Resonance (SPR): Provides real-time kinetic measurements of ST13-Hsc70 binding, determining association and dissociation rates under varying conditions (pH, temperature, nucleotide state).

• Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding, providing insights into the energetics of ST13-Hsc70 interactions.

• Co-immunoprecipitation from cellular extracts: Assesses interactions in a more native environment, especially when combined with mass spectrometry for identification of additional complex components.

These methods can be combined with mutational analysis of key residues in both ST13 and Hsc70 to map the precise interaction interface and understand how different domains contribute to complex formation and stability.

How can one design experiments to investigate the role of chicken ST13 in protein quality control?

Designing experiments to investigate ST13's role in protein quality control requires multi-faceted approaches:

• Cell-based degradation assays: Using reporter proteins prone to misfolding (e.g., mutant SOD1 or destabilized GFP variants) in cells with ST13 knockdown or overexpression to measure degradation kinetics.

• Reconstituted degradation systems: Combining purified components (ST13, Hsc70, CHIP E3 ligase, ubiquitin, proteasome) to reconstitute the protein quality control pathway in vitro and measure how ST13 affects substrate ubiquitination and degradation rates.

• Client specificity profiling: Employing techniques like the UBAIT system to identify which substrates are specifically affected by ST13 manipulation. Studies show that Hsc70 and Hsp70 have largely non-overlapping client specificities , and ST13 may influence this specificity.

• Stress response experiments: Exposing cells to various stressors (heat shock, oxidative stress, expression of aggregation-prone proteins) with and without ST13 manipulation to assess its role in stress resistance.

Data from search result suggests that Hsc70-4 and CHIP mediate the degradation of plastid-destined precursors, providing a specific system where ST13's role could be investigated. Similarly, research showing CHIP's role in tau degradation offers another model system for studying ST13 in protein quality control.

What are the critical controls needed when working with recombinant chicken ST13 in functional assays?

When conducting functional assays with recombinant chicken ST13, several critical controls must be implemented:

• Expression system controls:

  • Comparison with native chicken ST13 (if available) to assess functional differences

  • Analysis of post-translational modifications using mass spectrometry to account for system-specific PTMs

  • Evaluation of the same protein from different expression systems to disentangle PTM effects from intrinsic protein function

• Activity controls:

  • Mutant ST13 versions (e.g., binding-deficient mutants) to confirm specificity of observed effects

  • ATPase activity measurements of Hsc70 alone vs. with ST13 to confirm co-chaperone functionality

  • Thermal stability assays (DSF/DSC) to ensure properly folded protein

• Specificity controls:

  • Other co-chaperones (e.g., BAG family proteins) to distinguish ST13-specific effects from general co-chaperone effects

  • Cross-species Hsc70 proteins to determine specificity of chicken ST13 for avian vs. mammalian Hsc70

• Endogenous background control:

  • For cellular assays, CRISPR knockout or effective siRNA knockdown of endogenous ST13 to prevent interference

  • Validation of antibody specificity for distinguishing recombinant from endogenous protein

Research on DnaK demonstrates that expression system-induced PTMs significantly alter electrostatic properties and function , emphasizing the importance of proper controls when working with recombinant ST13.

What are common challenges in expressing and purifying functional recombinant chicken ST13?

Researchers face several technical challenges when working with recombinant chicken ST13:

• Solubility issues: ST13 can form inclusion bodies in bacterial expression systems, requiring optimization of:

  • Induction conditions (temperature, IPTG concentration)

  • Fusion tags (solubility enhancers like MBP or SUMO)

  • Co-expression with chaperones

• Post-translational modification heterogeneity: As shown in studies with DnaK , expression systems introduce variable PTMs that affect function. Researchers should consider:

  • Mass spectrometry analysis to characterize PTM patterns

  • Comparison of multiple expression systems

  • Phosphatase treatment to assess the impact of phosphorylation

• Protein stability challenges: ST13 may exhibit limited stability in solution, requiring:

  • Buffer optimization (pH, salt, additives like glycerol)

  • Storage condition testing (-80°C storage is recommended)

  • Avoidance of repeated freeze-thaw cycles

• Co-purification of endogenous chaperones: Bacterial chaperones may co-purify with ST13, necessitating:

  • Rigorous washing steps during affinity purification

  • Secondary purification methods

  • Quality control to ensure purity of final preparations

• Activity verification: Confirming that purified ST13 retains its co-chaperone activity requires:

  • Functional assays showing modulation of Hsc70 ATPase activity

  • Client binding assays

  • Structural verification (e.g., circular dichroism, thermal shift assays)

Addressing these challenges requires systematic optimization of expression and purification protocols specific to chicken ST13.

How can researchers overcome issues with recombinant chicken ST13 activity loss during experimental procedures?

Activity loss during experimental procedures is a common challenge that can be addressed through several methodological approaches:

• Stabilizing buffer formulations:

  • Include osmolytes (glycerol 5-10%, trehalose)

  • Optimize ionic strength (typically 150-300 mM NaCl)

  • Include reducing agents (DTT, β-mercaptoethanol) to prevent oxidation of cysteine residues

  • Consider chaperone stabilizing agents (ADP at low concentrations)

• Temperature management:

  • Maintain samples on ice when possible

  • Minimize incubation times at elevated temperatures

  • Pre-cool equipment and reagents

• Protein concentration effects:

  • Test activity at various protein concentrations (ST13 may aggregate at high concentrations)

  • Consider carrier proteins for very dilute solutions

• Storage optimization:

  • Aliquot protein to avoid repeated freeze-thaw cycles

  • Store at -80°C for long-term stability

  • Test flash-freezing in liquid nitrogen vs. gradual freezing

• Activity preservation additives:

  • BSA as a stabilizer (0.1-1 mg/ml)

  • Protease inhibitor cocktails

  • ATP/ADP to stabilize conformation

Evidence from studies with similar proteins suggests that co-chaperones like ST13 are sensitive to experimental conditions, and careful optimization is required to maintain their functional integrity throughout experimental procedures.

How can CRISPR-Cas9 technology be used to study chicken ST13 function in avian cell lines?

CRISPR-Cas9 technology offers powerful approaches for studying chicken ST13 function:

• Knockout studies: Complete elimination of ST13 expression to assess:

  • Effects on Hsc70 client processing

  • Changes in stress response pathways

  • Impacts on viral infection (particularly relevant given Hsp70's role in IBDV infection)

  • Alterations in protein quality control

• Domain-specific editing: Creating truncations or specific mutations to determine:

  • Which domains are essential for Hsc70 interaction

  • How different domains contribute to co-chaperone function

  • Separation of different functions (e.g., Hsc70 binding vs. client interaction)

• Endogenous tagging: Adding fluorescent or affinity tags to study:

  • Subcellular localization under different conditions

  • Dynamic protein-protein interactions using FRET or BiFC

  • Chromatin immunoprecipitation to identify potential transcriptional roles

• Inducible systems: Combining CRISPR with inducible promoters to:

  • Control timing of ST13 expression/deletion

  • Study acute vs. chronic effects of ST13 manipulation

  • Create conditional knockouts for essential functions

When designing CRISPR experiments, researchers should consider avian-specific factors such as optimal codon usage for guide RNA expression, efficiency of homology-directed repair in chicken cells, and appropriate control cell lines. The DF-1 cell line (immortalized chicken fibroblasts) used in IBDV studies represents a suitable model system for such experiments.

What are emerging technologies for studying the dynamic interactions of chicken ST13 in the chaperone network?

Several cutting-edge technologies are advancing our understanding of dynamic ST13 interactions:

• Proximity labeling approaches:

  • BioID and TurboID fusions with ST13 to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal resolution of interactions

  • The UBAIT system demonstrated for HSP70 client identification could be adapted for ST13

• Single-molecule techniques:

  • Fluorescence correlation spectroscopy (FCS) to measure binding kinetics in solution

  • Single-molecule FRET to observe conformational changes during interactions

  • Optical tweezers to measure forces involved in chaperone-assisted protein folding

• Structural biology advances:

  • Cryo-electron microscopy to visualize ST13-chaperone complexes

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

  • Integrative structural biology combining multiple data types

• Live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure ST13 dynamics

  • Optogenetic control of ST13 localization or activity

  • Super-resolution microscopy to visualize chaperone complexes below the diffraction limit

• Systems biology approaches:

  • Proteome-wide thermal profiling to identify clients affected by ST13

  • Network analysis of chaperone systems under different stress conditions

  • Integration of multiple -omics approaches to build comprehensive models

These technologies provide unprecedented insights into the spatial and temporal dynamics of ST13 within the chaperone network, facilitating a more complete understanding of its function in protein homeostasis.

How does chicken ST13 differ from its homologs in other species in terms of structure and function?

Comparative analysis of ST13 across species reveals evolutionary patterns that inform functional understanding:

Studies of Hsp70 homologs demonstrated that despite high sequence similarity, HSP70 and HSC70 have largely non-overlapping specificities . Similar principles may apply to ST13 homologs across species, with subtle sequence variations potentially resulting in significant functional differences that are adapted to species-specific proteostasis requirements.

What insights can be gained from studying chicken ST13 that may not be apparent from mammalian models?

Studying chicken ST13 offers several unique advantages and insights:

• Temperature adaptation mechanisms: Birds maintain higher body temperatures (40-42°C) than mammals, requiring adaptations in their chaperone systems. Chicken ST13 may have evolved specialized features to function optimally at these elevated temperatures.

• Avian-specific viral interactions: As demonstrated with IBDV, chicken chaperones play roles in avian-specific viral infections . Studying chicken ST13 can reveal how co-chaperones are involved in host-virus interactions that may differ from mammalian systems.

• Developmental biology insights: Birds undergo unique developmental processes (e.g., egg incubation with temperature fluctuations), potentially requiring specialized functions of the ST13-Hsc70 system during development.

• Evolutionary adaptations in protein quality control: Birds have evolved flight, which imposes high metabolic demands and potential protein damage from oxidative stress. Chicken ST13 may reveal adaptations in protein quality control systems that address these unique physiological challenges.

• Agricultural and veterinary applications: Understanding chicken ST13 has direct applications for poultry health and disease resistance, with potential for developing targeted interventions for avian-specific diseases.

These avian-specific insights complement knowledge gained from mammalian models, providing a more comprehensive understanding of chaperone system evolution and adaptation across vertebrates.

What are the most promising future research directions for chicken ST13 in avian biology?

Several research directions hold significant promise for advancing our understanding of chicken ST13:

• Systems-level analysis of the avian chaperone network: Comprehensive mapping of ST13 interactions within the chicken chaperone system using proteomics and interactomics approaches.

• Role in avian development: Investigation of ST13's function during key developmental stages, particularly during embryonic development and stress responses in ovo.

• Antiviral strategies targeting ST13-Hsc70 interactions: Building on findings that Hsp70 is essential for IBDV infection , exploring ST13 as a potential target for intervention in avian viral diseases.

• Comparative studies across avian species: Examining ST13 variation across diverse bird species to understand adaptations to different environmental niches and stress conditions.

• Integration of structural biology with functional studies: Obtaining high-resolution structures of chicken ST13 in complex with Hsc70 and clients to guide rational design of modulators.

• Agricultural applications: Developing genetic markers or biotechnological applications based on ST13 function to improve stress resistance in poultry.

These directions would significantly advance both fundamental understanding of protein quality control in avian biology and applied research in veterinary and agricultural sciences.

What methodological advances are needed to better understand chicken ST13 function in vivo?

Several methodological advances would significantly enhance our understanding of chicken ST13 function:

• Improved avian genetic models: Development of more sophisticated transgenic chicken models using advanced genome editing, including:

  • Conditional knockout systems

  • Tissue-specific promoters for targeted expression

  • Reporter knock-ins for endogenous protein visualization

• Advanced imaging for avian systems:

  • Adaptation of intravital imaging techniques for avian embryos

  • Development of avian-specific cell lines with fluorescent organelle markers

  • Implementation of tissue clearing methods optimized for avian tissues

• Avian-specific proteomics resources:

  • Comprehensive chicken interactome databases

  • Improved annotation of post-translational modifications in avian proteins

  • Specialized software for analyzing avian-specific protein interaction networks

• Functional genomics tools:

  • Avian-optimized CRISPR screening libraries

  • RNA-seq and ChIP-seq protocols optimized for avian samples

  • Single-cell technologies adapted for avian cells

• Ex vivo models:

  • Organoid systems derived from chicken tissues

  • Perfusion systems for studying ST13 function in isolated tissues

  • Primary cell culture systems that better maintain avian cellular characteristics

These methodological advances would bridge current gaps in our ability to study chicken ST13 function in physiologically relevant contexts, leading to more accurate understanding of its roles in avian biology.