Recombinant Danio rerio Ankyrin repeat domain-containing protein 13C (ankrd13c)

What is ANKRD13C and what is its functional role in zebrafish (Danio rerio)?

ANKRD13C is a protein characterized by ankyrin repeat domains that functions as a molecular chaperone for G protein-coupled receptors (GPCRs). In zebrafish and other organisms, it regulates the folding and maturation of newly synthesized GPCRs . This protein is primarily associated with the cytosolic side of endoplasmic reticulum (ER) membranes, where it interacts with the cytoplasmic C-terminus of GPCRs .

The functional significance of ANKRD13C includes:

• Promotion of GPCR biogenesis by inhibiting degradation of newly synthesized receptors

• Regulation of protein exit from the ER

• Control of protein trafficking through the biosynthetic pathway

• Retention of misfolded/unassembled forms of receptors in the ER, directing them to proteasome-mediated degradation

Research methodology for functional characterization typically involves protein interaction studies, subcellular localization experiments, and functional assays measuring receptor expression levels under conditions of ANKRD13C overexpression or knockdown.

How is the structure of zebrafish ANKRD13C characterized and what are its key domains?

Zebrafish ANKRD13C is a 488 amino acid protein that contains multiple functional domains . While the search results don't provide the complete structural characterization specifically for zebrafish ANKRD13C, we can infer from related ankyrin repeat domain-containing proteins that the structure likely includes:

• Ankyrin repeat domains - critical for protein-protein interactions

• Potential PEST sequences - involved in rapid intracellular proteolysis

• Possible coiled-coil motifs - important for protein oligomerization

• Nuclear localization signals (NLS) may be present or absent depending on specific function

The full amino acid sequence begins with: MTGEKIRSVR KERKSGLDLL EPDEEPAATG PAKHRGSKIF SGGNHRISRS SSSPGDPDGA YP...

Methodological approaches to study ANKRD13C structure include recombinant protein expression (typically in yeast systems for eukaryotic proteins), followed by purification and structural analysis through techniques such as X-ray crystallography or NMR spectroscopy.

What expression patterns does ANKRD13C show during zebrafish development?

While the search results don't provide specific developmental expression data for ANKRD13C in zebrafish, we can gain insights from the expression patterns of related ankyrin repeat domain-containing proteins. For example, ankrd1a and ankrd1b (counterparts of mammalian ANKRD1) show distinct spatiotemporal expression during development:

• ankrd1a shows mild increase at 72 hpf (1.74±0.24 fold increase relative to 24 hpf)

• ankrd1b is markedly upregulated from 24 hpf onward, peaking at 72 hpf (92.18±36.95 fold increase relative to 24 hpf)

• These paralogs exhibit non-overlapping expression patterns during skeletal muscle development, with ankrd1a predominantly in trunk somites and ankrd1b in tail somites

To study ANKRD13C expression patterns, researchers should employ:

• Quantitative PCR (qPCR) with appropriate reference genes (e.g., rpl13a as used for other ankyrin repeat proteins)

• In situ hybridization to visualize spatial expression

• Developmental time-course experiments from early embryogenesis through adult stages

What are the optimal conditions for expressing and purifying recombinant zebrafish ANKRD13C?

For optimal expression of recombinant zebrafish ANKRD13C, the following methodological approach is recommended:

Expression System Selection:
The yeast expression system is particularly suitable for zebrafish ANKRD13C as it combines economic efficiency with the eukaryotic post-translational modifications necessary for proper protein folding . This system enables modifications such as glycosylation, acylation, and phosphorylation that ensure native protein conformation.

Protein Characteristics for Optimal Expression:

• Full-length construct: Amino acids 1-488

• Addition of purification tag: His-tag for affinity purification

• Expression vector: Choose vectors optimized for yeast expression with inducible promoters

Purification Protocol:

• Cell lysis under non-denaturing conditions to preserve protein structure

• Affinity chromatography using Ni-NTA columns for His-tagged proteins

• Size exclusion chromatography for further purification

• Quality control through SDS-PAGE and Western blotting

• Lyophilization for storage while maintaining activity

Researchers should aim for >90% purity for experimental applications . Alternative expression systems (E. coli, mammalian cells, or baculovirus infection) may be considered depending on specific experimental requirements, though these have trade-offs in terms of cost, yield, and post-translational modifications.

How does ANKRD13C interact with G protein-coupled receptors in cellular models?

ANKRD13C functions as a molecular chaperone for GPCRs, with several key interaction mechanisms identified through cellular studies:

Interaction Mechanism:

• ANKRD13C directly binds to the cytoplasmic C-terminus of GPCRs

• This interaction occurs at the endoplasmic reticulum membrane on the cytosolic side

• The binding shows specificity toward GPCRs, as ANKRD13C does not affect the expression of unrelated proteins like GFP, GRK2, or VSVG

Functional Consequences of Interaction:

• Initial increase in receptor protein levels upon co-expression

• Inhibition of degradation of newly synthesized receptors

• Prolonged interaction results in ER retention of misfolded/unassembled receptors

• Direction of misfolded receptors to proteasome-mediated degradation

Experimental Approaches to Study Interactions:

• Co-immunoprecipitation to detect physical interaction

• Yeast two-hybrid screening (which originally identified ANKRD13C as a GPCR-interacting protein)

• Fluorescence microscopy showing co-localization with ER markers

• Pulse-chase experiments to track receptor biogenesis and degradation

• siRNA knockdown studies to assess effects of reduced ANKRD13C expression

What methodologies are most effective for studying ANKRD13C function in zebrafish models?

In Vivo Zebrafish Model Preparation:

• Select adult zebrafish (both sexes, aged 60-90 days, sizes 3.5 ± 0.5 cm and weight 0.4 ± 0.1 g)

• Maintain in appropriate conditions: glass aquariums (30 x 15 x 20 cm), dechlorinated water with air pumps and submerged filters, at 25°C and pH 7.0, with a 14-day circadian cycle (10 h light/dark)

• Acclimate for at least 24 hours before experiments

Gene Expression Analysis:

• Quantitative PCR (qPCR) using appropriate reference genes (e.g., rpl13a)

• Reaction conditions: initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, annealing and elongation at 60°C

• Data analysis using the 2^(-ΔΔCt) method for relative quantification

Genetic Manipulation Approaches:

• Morpholino knockdown for transient loss-of-function

• CRISPR/Cas9 for generating stable genetic models

• Transgenic overexpression using tissue-specific promoters

Protein Analysis Techniques:

• Western blotting with specific antibodies

• Immunohistochemistry for tissue localization

• Co-immunoprecipitation for protein interaction studies

Functional Studies:

• Tissue-specific expression analysis in response to physiological stimuli

• Exercise protocols to study stress responses (e.g., forced swimming for 3 hours, twice daily, for five consecutive days)

• Behavioral assays to assess phenotypic outcomes

How does environmental stress impact ANKRD13C expression and function in zebrafish?

While direct data on ANKRD13C responsiveness to stress is limited in the search results, insights can be drawn from related ankyrin repeat proteins. The effect of physical stress (endurance exercise) on ankyrin repeat proteins in zebrafish has been documented:

Exercise Protocol Impact on Ankyrin Repeat Proteins:

• Protocol: Two 3-hour bouts of forced swimming daily for five consecutive days

• Tissue-specific responses measured 3 hours after the final exercise bout:

  • ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change)

  • ankrd1b expression increased in skeletal muscle (1.97±1.05 fold change)

  • ankrd2 expression increased in skeletal muscle (1.84±0.58 fold change)

Methodological Approach for Studying Stress Responses:

• Design appropriate stress models (e.g., exercise, oxidative stress, temperature variation)

• Tissue sampling at different time points post-stress

• Gene expression analysis via qPCR with reference genes like rpl13a

• Protein analysis through Western blotting

• Subcellular localization studies using immunofluorescence

Based on the function of ankyrin repeat proteins as stress-responsive elements, researchers should consider examining ANKRD13C in the context of:

• Mechanical stress responses in muscle tissue

• Oxidative stress conditions

• Metabolic stress scenarios

• Temperature stress paradigms

What are the key controls needed when working with recombinant zebrafish ANKRD13C?

When designing experiments with recombinant zebrafish ANKRD13C, researchers should implement the following controls:

For Expression Studies:

• Empty vector controls to account for expression system effects

• Unrelated protein controls (e.g., GFP) to demonstrate specificity

• Wild-type vs. mutated ANKRD13C to identify functional domains

• Dose-response experiments to determine optimal protein concentrations

For Interaction Studies:

• Non-GPCR membrane proteins as negative controls

• Multiple GPCR subtypes to assess interaction specificity

• Truncated ANKRD13C constructs lacking specific domains

• Competition assays with known interacting proteins

For Functional Assays:

• Proteasome inhibitors (to confirm degradation pathways)

• ER stress inducers to assess role in stress response

• Pulse-chase experiments with and without ANKRD13C expression

• siRNA knockdown with rescue experiments using recombinant protein

For In Vivo Studies:

• Age-matched and sex-balanced zebrafish groups

• Appropriate reference genes for qPCR (e.g., rpl13a)

• Vehicle-only treatments in parallel with experimental conditions

• Behavioral monitoring to assess systemic effects

How can researchers effectively compare zebrafish ANKRD13C with its mammalian orthologs?

To systematically compare zebrafish ANKRD13C with mammalian orthologs, researchers should implement the following methodological approaches:

Sequence and Structural Analysis:

• Multiple sequence alignment to identify conserved domains

• Phylogenetic analysis to understand evolutionary relationships

• Synteny analysis to examine genomic context conservation

• Protein structure prediction and comparison

Based on related ankyrin repeat proteins, expect moderate sequence conservation between species, with higher conservation in functional domains like ankyrin repeats . For example, zebrafish Ankrd1a, Ankrd1b, and Ankrd2 show 56%, 46%, and 51% identity with their human counterparts, respectively .

Functional Comparison:

• Parallel expression studies in zebrafish and mammalian cells

• Cross-species rescue experiments

• GPCR interaction profiles across species

• Comparative response to cellular stressors

Experimental Design Table for Cross-Species Comparison:

How should researchers interpret conflicting data from different ANKRD13C expression systems?

When facing conflicting results from different expression systems for zebrafish ANKRD13C, researchers should systematically evaluate:

Source of Variation Analysis:

• Expression system differences: Yeast expression provides eukaryotic post-translational modifications that may be critical for proper ANKRD13C folding and function, while bacterial systems may yield higher amounts but lack these modifications

• Protein tag influence: His-tag location (N- vs C-terminal) may differentially affect protein function

• Protein purity variations: Aim for >90% purity for consistent results

• Buffer composition effects on protein stability and activity

Methodological Approach to Resolve Conflicts:

• Side-by-side comparison of proteins from different expression systems using multiple functional assays

• Structural analysis to identify potential differences in protein folding

• Mass spectrometry to confirm post-translational modifications

• Limited proteolysis to assess protein conformation

Decision Table for Expression System Selection:

What are common pitfalls in ANKRD13C research and how can they be avoided?

Common Experimental Challenges and Solutions:

• Low Protein Expression Yields

  • Problem: Recombinant ANKRD13C may express poorly in certain systems

  • Solution: Optimize codon usage for expression system, adjust induction conditions, consider fusion partners to enhance solubility

• Protein Aggregation

  • Problem: ANKRD13C may form aggregates during expression or purification

  • Solution: Express at lower temperatures, include stabilizing agents in buffers, consider detergents for membrane-associated portions

• Non-specific Interactions

  • Problem: His-tagged proteins may show non-specific binding in interaction studies

  • Solution: Include imidazole controls, perform reciprocal co-IP, validate with multiple detection methods

• Inconsistent qPCR Results

  • Problem: Variable reference gene stability across experimental conditions

  • Solution: Validate reference genes (like rpl13a) for specific experimental conditions , use multiple reference genes

• Developmental Variability

  • Problem: Expression timing may vary between zebrafish clutches

  • Solution: Precise staging of embryos, larger sample sizes, consistent husbandry conditions

Methodological Controls to Implement:

• Include wild-type controls alongside recombinant protein experiments

• Perform dose-response studies to identify optimal protein concentrations

• Validate antibody specificity through knockdown/knockout controls

• Include positive controls with known behavior in each experimental set

What emerging technologies show promise for advancing ANKRD13C research?

Several cutting-edge technologies hold significant potential for advancing our understanding of zebrafish ANKRD13C:

CRISPR/Cas9 Genome Editing:

• Generate precise knockouts or domain-specific mutations in zebrafish ANKRD13C

• Create fluorescent protein fusions at endogenous loci for live imaging

• Implement tissue-specific or inducible CRISPR systems for temporal control

Advanced Imaging Approaches:

• Super-resolution microscopy to visualize ANKRD13C-GPCR interactions at the nanoscale

• Live cell imaging with optogenetic tools to manipulate ANKRD13C activity

• Light-sheet microscopy for whole-organism visualization of ANKRD13C dynamics

Proteomics Applications:

• Proximity labeling (BioID, APEX) to identify the complete ANKRD13C interactome

• Quantitative proteomics to measure changes in the proteome upon ANKRD13C manipulation

• Cross-linking mass spectrometry to map interaction interfaces

Computational Methods:

• Molecular docking studies to predict ANKRD13C-GPCR binding, similar to approaches used for other proteins in zebrafish

• AlphaFold or RoseTTAFold for accurate structure prediction

• Network analysis to position ANKRD13C within cellular signaling pathways

Single-Cell Technologies:

• scRNA-seq to identify cell populations expressing ANKRD13C during development

• Spatial transcriptomics to map expression patterns with tissue context

• Cell-specific proteomics to measure ANKRD13C levels across different cell types

How might ANKRD13C research contribute to broader understanding of protein quality control mechanisms?

Research on zebrafish ANKRD13C offers valuable insights into fundamental aspects of protein quality control:

Evolutionary Conservation of Chaperone Functions:

• Comparing ANKRD13C function across species can reveal evolutionarily conserved mechanisms of protein quality control

• The moderate conservation observed in related ankyrin repeat proteins (46-56% identity between zebrafish and human) suggests both conserved and divergent aspects of function

GPCR-Specific Quality Control:

• ANKRD13C selectively regulates GPCR biogenesis and trafficking, distinguishing it from general chaperones

• Research can reveal how specialized chaperones recognize specific protein families

• Understanding this selectivity may lead to targeted therapeutic approaches for GPCR-related diseases

ER-Associated Degradation (ERAD) Mechanisms:

• ANKRD13C directs misfolded GPCRs to proteasomal degradation

• This provides a model system to study how quality control decisions (fold vs. degrade) are made

• The balance between retention, folding assistance, and degradation reveals fundamental principles of proteostasis

Stress Response Integration:

• Related ankyrin repeat proteins respond to exercise stress in zebrafish

• ANKRD13C may similarly integrate stress signals into quality control decisions

• This connects environmental inputs to cellular protein homeostasis mechanisms