Recombinant Danio rerio Ankyrin repeat domain-containing protein 46 (ankrd46)

How does the structure of Danio rerio ankrd46 compare to other ankyrin repeat domain-containing proteins?

Danio rerio ankrd46 belongs to the ankyrin repeat protein family, characterized by the presence of ankyrin repeat motifs that are crucial for protein-protein interactions. While specific structural data for ankrd46 is limited, comparison with other ankyrin repeat proteins in zebrafish shows certain conserved features:

Phylogenetic analysis of ankyrin repeat proteins shows distinct evolutionary relationships, with specific clustering patterns that reflect functional similarities. For example, in zebrafish, Ankrd1a and Ankrd1b are paralogous proteins that form a distinct cluster separate from Ankrd2 .

What are the recommended storage and reconstitution protocols for recombinant ankrd46 protein?

For optimal stability and activity of recombinant ankrd46 protein, the following storage and reconstitution protocols are recommended:

Storage Recommendations:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Avoid repeated freezing and thawing as this may affect protein stability and activity

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C

• The recommended default final concentration of glycerol is 50%

The protein is stored in Tris/PBS-based buffer containing 6% Trehalose, pH 8.0, which helps maintain stability during storage .

How are ankrd46 and related ankyrin repeat proteins expressed during zebrafish development?

While specific expression data for ankrd46 during zebrafish development is limited in the provided search results, insights can be gained from the expression patterns of related ankyrin repeat domain-containing proteins:

Developmental Expression Profiles:

• ankrd1a: Shows mild increase in expression at 72 hours post fertilization (hpf), with approximately 1.74±0.24 fold increase relative to the 24 hpf time point .

• ankrd1b: Exhibits markedly different expression dynamics with significant upregulation from 24 hpf onward, peaking at 72 hpf with a dramatic 92.18±36.95 fold increase relative to 24 hpf .

• ankrd2: Expression is barely detectable during early development stages .

Spatial Expression Patterns:

• The paralogous genes ankrd1a and ankrd1b show non-overlapping expression patterns during skeletal muscle development, with ankrd1a predominantly expressed in trunk somites and ankrd1b in tail somites .

These differential spatiotemporal expression patterns suggest specific roles for different ankyrin repeat proteins during zebrafish development, with precise regulation according to developmental stage and tissue type. It is reasonable to hypothesize that ankrd46 may also exhibit tissue-specific and developmentally regulated expression patterns that correspond to its functional role.

What techniques are most effective for detecting ankrd46 expression in zebrafish tissues?

Based on methodologies successfully employed for studying related ankyrin repeat proteins, the following techniques are recommended for detecting ankrd46 expression in zebrafish tissues:

1. Quantitative Real-Time PCR (qPCR):

• RNA isolation from embryos or adult tissues using Trizol reagent

• DNase I treatment to eliminate genomic DNA contamination

• cDNA synthesis followed by qPCR with gene-specific primers

• Normalization to reference genes (e.g., rpl13a) that show stable expression across developmental stages and experimental conditions

• Analysis using the 2(-ΔΔCt) method

2. Whole-Mount In Situ Hybridization (ISH):

• Generation of specific RNA probes by PCR amplification from cDNA

• Cloning into appropriate vectors (e.g., pGEM-T easy)

• Synthesis of labeled RNA probes using appropriate RNA polymerases and digoxigenin-labeled nucleotides

• Hybridization at 67°C overnight with approximately 300 ng of probe

• For embryos older than 24 hpf, treatment with 0.003% 1-phenyl 2-thiourea (PTU) to prevent pigmentation

3. Protein Detection Methods:

• Western blotting with specific antibodies

• Immunohistochemistry for tissue localization

• Fluorescent protein tagging for live imaging studies

When designing primers for ankrd46 detection, it's crucial to ensure specificity by targeting unique regions that don't cross-react with other ankyrin repeat family members. The high sequence similarity between some family members necessitates careful primer design and validation.

How does exercise training affect ankrd46 expression in zebrafish, and what protocols are effective for such studies?

While the direct effect of exercise on ankrd46 specifically isn't detailed in the search results, data from related ankyrin repeat proteins provides valuable insights and methodological approaches:

Exercise Effects on Related Proteins:

• Endurance exercise training induces differential upregulation of ankyrin repeat proteins in zebrafish striated muscle

• After five days of exercise training, ankrd1a expression increases in cardiac muscle (6.19±5.05 fold change)

• ankrd1b and ankrd2 are upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively)

Recommended Exercise Protocol for Studying ankrd46:

• Setup: Use a 5L glass beaker with a 60x10 mm stir bar, filled with 4L of fish water and placed on a magnetic stirrer

• Adaptation: Pre-exercise fish for two days, 3 hours per day, with stirrer speed adjusted to generate a 2 cm deep vortex

• Exercise regimen: Increase rotation speed to generate a 10 cm deep vortex and subject fish to two sets of 3 hours swimming, with 1 hour resting and feeding in between, for 5 consecutive days

• Tissue collection: Harvest organs 3 hours after the last exercise bout

• Sample preparation: Collect skeletal muscles individually or pooled by two, and pool two hearts in each cardiac sample

This protocol has been validated for studying exercise-induced changes in gene expression in zebrafish and should be applicable for investigating ankrd46 responses to increased muscle activity. It provides a standardized approach to induce physiological adaptations that may alter ankrd46 expression.

What are the recommended methods for overexpression or knockdown of ankrd46 in zebrafish models?

For effective manipulation of ankrd46 expression in zebrafish, several approaches can be employed:

Overexpression Methods:

• mRNA Microinjection:

  • Clone the full-length ankrd46 coding sequence into an appropriate expression vector

  • Linearize the plasmid and synthesize capped mRNA using mMESSAGE mMACHINE kits

  • Inject synthesized mRNA (typically 50-200 pg) into one-cell stage embryos

  • Include appropriate controls (e.g., GFP mRNA)

• Tol2 Transposon-Mediated Transgenesis:

  • Clone ankrd46 into a Tol2 transposon-containing vector with a tissue-specific promoter

  • Co-inject with Tol2 transposase mRNA into one-cell stage embryos

  • Screen for successful integration and expression

Knockdown/Knockout Methods:

• Morpholino Oligonucleotides:

  • Design translation-blocking or splice-blocking morpholinos targeting ankrd46

  • Inject 1-8 ng morpholino into one-cell stage embryos

  • Include standard control morpholino and perform rescue experiments to validate specificity

• CRISPR/Cas9 Gene Editing:

  • Design guide RNAs targeting conserved exons of ankrd46

  • Co-inject guide RNA with Cas9 mRNA or protein

  • Screen F0 embryos for mutations using high-resolution melt analysis or T7 endonuclease assay

  • Raise potential founders to establish stable mutant lines

Validation Approaches:

• Confirm successful overexpression or knockdown using qPCR and Western blotting

• Assess phenotypic changes through morphological examination and functional assays

• Perform RNA-seq to identify downstream effects on gene expression patterns

These approaches allow for both transient and stable manipulation of ankrd46 expression in zebrafish, facilitating comprehensive functional characterization in different developmental stages and tissue contexts.

What protein-protein interactions have been identified for ankrd46, and how can these interactions be experimentally validated?

While specific protein-protein interactions for ankrd46 are not directly described in the search results, its structural features as an ankyrin repeat domain-containing protein suggest a role in protein-protein interactions. Based on knowledge of related proteins, several approaches can be employed to identify and validate ankrd46 interaction partners:

Potential Interaction Domains:

• The ankyrin repeat domains in ankrd46 are likely critical for protein-protein interactions, as these motifs are known to mediate such interactions in other family members

• PEST sequences may regulate protein stability and interactions with the protein degradation machinery

Methods for Identifying Interaction Partners:

• Yeast Two-Hybrid Screening:

  • Use full-length ankrd46 or specific domains as bait

  • Screen against zebrafish cDNA libraries

  • Validate positive interactions through secondary screens

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of ankrd46 (e.g., His-tagged as described in )

  • Perform pulldown experiments followed by mass spectrometry

  • Validate specific interactions with candidate proteins

• Proximity Labeling Approaches:

  • Generate BioID or APEX2 fusions with ankrd46

  • Express in zebrafish cells or tissues

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

Validation Strategies:

• Reciprocal Co-IP:

  • Perform pulldowns with both ankrd46 and identified partners

  • Confirm interactions in both directions

• Domain Mapping:

  • Generate truncation mutants to identify specific interaction domains

  • Focus on ankyrin repeat regions (amino acids ~30-130 based on related proteins)

• Functional Validation:

  • Co-express ankrd46 and interaction partners in zebrafish

  • Assess effects on localization, expression, and function

  • Perform knockdown/knockout studies to examine dependency relationships

These approaches would provide comprehensive insights into the protein interaction network of ankrd46, enhancing our understanding of its functional role in zebrafish development and physiology.

How can researchers distinguish between the functions of ankrd46 and other ankyrin repeat domain-containing proteins in zebrafish?

Distinguishing the specific functions of ankrd46 from other ankyrin repeat domain-containing proteins requires careful experimental design:

Comparative Analysis Approaches:

• Expression Pattern Comparison:

  • Perform side-by-side in situ hybridization for ankrd46 and related proteins

  • Compare spatiotemporal expression patterns during development

  • Identify unique versus overlapping expression domains

  Related ankyrin repeat proteins like ankrd1a and ankrd1b show non-overlapping expression patterns (trunk vs. tail somites), suggesting distinct functions .

• Response to Physiological Stimuli:

  • Expose zebrafish to various stimuli (e.g., exercise as detailed in section 3.1)

  • Compare expression changes across different ankyrin repeat family members

  • Identify differential responses that suggest unique functions

  For example, ankrd1a responds primarily in cardiac muscle while ankrd1b and ankrd2 are upregulated in skeletal muscle following exercise .

• Specific Loss-of-Function Studies:

  • Generate specific knockdown/knockout models for each family member

  • Compare phenotypes to identify unique versus redundant functions

  • Perform rescue experiments with different family members to test functional equivalence

• Domain Swap Experiments:

  • Create chimeric proteins by swapping domains between ankrd46 and other family members

  • Express in zebrafish to determine which domains confer specific functions

  • Focus on ankyrin repeat domains and other functional motifs

Technical Considerations:

• Ensure Reagent Specificity:

  • Design highly specific primers/probes for qPCR and in situ hybridization

  • Validate antibody specificity using knockout controls

  • Use tagged proteins when antibody cross-reactivity is a concern

• Combinatorial Knockdown/Knockout:

  • Generate single and multiple knockouts of ankyrin repeat family members

  • Assess additive, synergistic, or redundant effects

  • Create conditional knockouts to circumvent early developmental lethality if present

These approaches, when systematically applied, will help delineate the specific functions of ankrd46 in relation to other family members, providing insights into both unique and overlapping roles.

How is ankrd46 evolutionarily conserved across species, and what does this suggest about its function?

Evolutionary conservation analysis of ankrd46 and related ankyrin repeat domain-containing proteins provides valuable insights into their functional significance:

Sequence Conservation:
While specific data for ankrd46 conservation isn't provided in the search results, related ankyrin repeat proteins in zebrafish show moderate conservation at the protein level when compared to mammalian orthologs. This conservation is more pronounced in the regions containing ankyrin repeats, highlighting the functional importance of these domains .

Conservation Table for Related Ankyrin Repeat Proteins:

Note: Zebrafish Ankrd1a and Ankrd1b show 47% identity to each other .

Functional Domain Conservation:

• Ankyrin repeats are conserved across species, reflecting their critical role in protein-protein interactions

• PEST sequences for protein degradation are found in all zebrafish MARPs

• Protein oligomerization motifs (coiled coils) show variable conservation, present in some family members (e.g., Ankrd1a) but not others (e.g., Ankrd1b and Ankrd2)

• Nuclear localization signals (NLS) are not universally conserved (e.g., absent in Ankrd1b)

Phylogenetic Relationships:
Phylogenetic analysis segregates ankyrin repeat proteins into distinct evolutionary groups. For example, ANKRD1 and ANKRD2 homologs form separate clusters, with teleost-specific Ankrd1b proteins closely related to Ankrd1a .

Functional Implications:
The pattern of evolutionary conservation suggests:

• The core protein-protein interaction function mediated by ankyrin repeats is fundamental and evolutionarily ancient

• Variable conservation of regulatory domains (NLS, coiled coils) indicates species-specific adaptations in regulation and localization

• Paralogous genes in zebrafish (like ankrd1a and ankrd1b) likely resulted from teleost-specific genome duplication, potentially leading to subfunctionalization

This evolutionary analysis provides a framework for understanding potential ankrd46 functions based on conserved domains and phylogenetic relationships, guiding functional studies across species.

How do zebrafish ankrd46 studies translate to understanding human ANKRD46 function and disease associations?

Zebrafish studies of ankrd46 can provide valuable insights into human ANKRD46 function and disease associations through comparative approaches:

Comparative Genetic Analysis:

• Human ANKRD46 has been associated with certain diseases through text mining of biomedical literature and database annotations , although specific disease details are not provided in the search results.

• Zebrafish ankrd46 studies can help elucidate the underlying molecular mechanisms that might explain these disease associations.

Translational Research Framework:

• Functional Conservation Assessment:

  • Compare protein domains, motifs, and interaction partners between zebrafish ankrd46 and human ANKRD46

  • Determine whether human ANKRD46 can rescue zebrafish ankrd46 knockdown/knockout phenotypes

  • Identify conserved regulatory pathways controlling expression

• Disease Modeling:

  • Introduce disease-associated mutations found in human ANKRD46 into zebrafish ankrd46

  • Assess phenotypic consequences at cellular and organismal levels

  • Test potential therapeutic approaches in the zebrafish model

• Expression Pattern Comparison:

  • Compare tissue-specific expression patterns between species

  • Identify conserved expression in disease-relevant tissues

  • Examine response to stress conditions that mimic disease states

Advantages of Zebrafish Models:

• Optical transparency allowing real-time visualization of developmental processes

• Genetic tractability for rapid generation of disease models

• High-throughput screening capability for drug discovery

• Cost-effective compared to mammalian models

• Well-characterized development enabling detailed phenotypic analysis

Methodological Approach:

• Generate ankrd46 zebrafish mutants using CRISPR/Cas9

• Characterize phenotypes at molecular, cellular, and organismal levels

• Perform rescue experiments with human ANKRD46

• Identify small molecules that modify phenotypes for potential therapeutic development

By leveraging the experimental advantages of zebrafish while maintaining focus on evolutionarily conserved aspects of ankrd46/ANKRD46 function, researchers can develop insights that meaningfully translate to human health and disease.

What are the critical factors for successful expression and purification of recombinant ankrd46 protein?

Successful expression and purification of recombinant ankrd46 protein requires attention to several critical factors:

Expression System Optimization:

• Host Selection: E. coli is commonly used for expressing recombinant ankrd46 protein . Consider BL21(DE3) or Rosetta strains for enhanced expression of eukaryotic proteins.

• Expression Vector: Use vectors with strong inducible promoters (T7, tac) and appropriate tag position. N-terminal His tags are commonly used for ankrd46 expression .

• Induction Conditions:

  • Temperature: Lower temperatures (16-25°C) often improve solubility

  • Inducer concentration: Optimize IPTG concentration (typically 0.1-1.0 mM)

  • Induction time: Test various durations (4-24 hours)

Purification Protocol Optimization:

• Lysis Conditions:

  • Buffer composition: Tris/PBS-based buffers at pH 8.0 are suitable

  • Include protease inhibitors to prevent degradation

  • Consider adding low concentrations of non-ionic detergents for membrane-associated variants

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged ankrd46

  • Consider secondary purification steps (ion exchange, size exclusion) for higher purity

  • Aim for >90% purity as determined by SDS-PAGE

• Quality Control:

  • Verify protein identity using mass spectrometry

  • Assess purity by SDS-PAGE (should exceed 90%)

  • Confirm proper folding with circular dichroism or limited proteolysis

Storage and Stability:

• Lyophilization: For long-term storage, lyophilization is recommended

• Buffer Components: Include stabilizers like trehalose (6%) in storage buffer

• Aliquoting: Prepare small aliquots to avoid freeze-thaw cycles

Troubleshooting Common Issues:

• Low Expression:

  • Check codon optimization for zebrafish protein in E. coli

  • Consider expressing as fusion with solubility enhancers (MBP, SUMO)

  • Test different growth media and cell densities at induction

• Poor Solubility:

  • Modify buffer conditions (salt concentration, pH)

  • Add solubilizing agents (glycerol, mild detergents)

  • Consider expressing truncated constructs focusing on stable domains

• Degradation:

  • Increase protease inhibitor concentration

  • Purify at lower temperatures (4°C)

  • Reduce purification time with optimized protocols

Following these recommendations should enable successful expression and purification of functional recombinant ankrd46 protein suitable for downstream applications.

How can researchers troubleshoot inconsistent results in ankrd46 expression studies?

Inconsistent results in ankrd46 expression studies can stem from multiple sources. Here's a systematic approach to identify and address common issues:

Sample Preparation Variables:

• RNA Quality:

  • Assess RNA integrity using bioanalyzer or gel electrophoresis

  • Ensure consistent RNA purity (A260/A280 ratio of 1.8-2.0)

  • Standardize DNase I treatment to eliminate genomic DNA contamination

• Tissue Collection:

  • Standardize dissection techniques

  • Minimize time between euthanasia and tissue collection

  • Consider flash freezing samples in liquid nitrogen

  • For pooled samples, maintain consistent ratios (e.g., two hearts per sample)

Technical Variables:

• qPCR Optimization:

  • Validate primer efficiency (should be 90-110%)

  • Perform technical triplicates for each sample

  • Include no-template and no-RT controls

  • Standardize threshold settings across experiments

• Reference Gene Selection:

  • Verify stability of reference genes across experimental conditions

  • Consider using multiple reference genes for normalization

  • The rpl13a gene has been validated as stable during development and exercise experiments

• In Situ Hybridization Consistency:

  • Standardize probe concentration (approximately 300 ng)

  • Maintain consistent hybridization temperature (67°C recommended)

  • Ensure proper PTU treatment for embryos older than 24 hpf to prevent pigmentation

Biological Variables:

• Developmental Timing:

  • Precisely stage embryos

  • Collect samples at consistent time points

  • Account for potential batch effects

• Exercise Protocol Standardization:

  • Maintain consistent vortex depth (10 cm for exercise condition)

  • Standardize exercise duration (two 3-hour sessions daily)

  • Control recovery period before sample collection (3 hours post-exercise)

Troubleshooting Checklist:

Validation Approaches:

• Use multiple detection methods (qPCR, in situ hybridization, Western blot)

• Include positive controls (known expressed genes like myod1 for muscle)

• Perform biological replicates from independent experiments

• Consider alternative normalization strategies

What are the most promising research areas for understanding ankrd46 function in zebrafish development and disease models?

Based on current knowledge of ankrd46 and related ankyrin repeat proteins, several promising research directions emerge:

Developmental Regulation and Patterning:

• Spatiotemporal Mapping: Comprehensive characterization of ankrd46 expression throughout zebrafish development using transgenic reporter lines and time-lapse imaging.

• Regulatory Networks: Identification of transcription factors and signaling pathways that control ankrd46 expression during development, potentially revealing how it integrates into broader developmental programs.

• Functional Role in Tissue Patterning: Investigation of ankrd46 involvement in specific developmental processes, particularly in tissues where related ankyrin repeat proteins show expression (e.g., somites, cardiac tissue) .

Stress Response and Adaptation:

• Exercise Physiology: Building on findings that related ankyrin repeat proteins respond to exercise , examination of ankrd46 regulation during different exercise protocols and training regimens.

• Tissue Regeneration: Investigation of ankrd46 role in zebrafish's remarkable regenerative capacity, particularly in tissues where ankyrin repeat proteins are expressed.

• Stress Signaling Integration: Exploration of how ankrd46 might participate in cellular stress responses, potentially connecting mechanical or metabolic stress to adaptive gene expression programs.

Disease Modeling:

• Cardiac Pathologies: Given the cardiac expression of related proteins , investigation of ankrd46 in zebrafish models of cardiac disease and heart failure.

• Skeletal Muscle Disorders: Exploration of ankrd46 function in models of muscular dystrophy or other muscle pathologies.

• Human Disease Variants: Introduction of mutations corresponding to human ANKRD46 variants associated with disease to create zebrafish models for mechanistic studies.

Molecular Interaction Networks:

• Protein Complex Identification: Comprehensive characterization of ankrd46 interaction partners using proteomics approaches.

• Subcellular Localization Dynamics: Investigation of ankrd46 trafficking between cellular compartments under different conditions.

• Post-translational Modifications: Characterization of how phosphorylation or other modifications regulate ankrd46 function and interactions.

These research directions would significantly advance our understanding of ankrd46 biology while leveraging the unique advantages of the zebrafish model system for in vivo studies of development, physiology, and disease.

What emerging technologies and methodologies could advance ankrd46 research in zebrafish models?

Several cutting-edge technologies and methodologies hold promise for advancing ankrd46 research in zebrafish:

Advanced Genome Editing Approaches:

• Base Editing and Prime Editing: These technologies allow precise nucleotide changes without double-strand breaks, enabling introduction of specific mutations corresponding to human variants.

• Conditional/Inducible CRISPR Systems: Tools like CreERT2-loxP or doxycycline-inducible Cas9 allow temporal and tissue-specific gene modification, overcoming potential early lethality of constitutive knockouts.

• CRISPR Screening: Pooled or arrayed CRISPR screens targeting potential interactors or regulators of ankrd46 to identify genetic networks.

Advanced Imaging Technologies:

• Light Sheet Microscopy: Allows long-term in vivo imaging with minimal phototoxicity, ideal for tracking ankrd46 expression and subcellular localization during development.

• Super-Resolution Microscopy: Techniques like STORM or PALM enable visualization of protein complexes at nanometer resolution, revealing detailed spatial organization of ankrd46 and its interaction partners.

• Correlative Light and Electron Microscopy (CLEM): Combines fluorescence imaging with ultrastructural analysis to place ankrd46 in precise cellular contexts.

Single-Cell Technologies:

• Single-Cell RNA-Seq: Reveals cell type-specific expression patterns and responses to perturbations, capturing heterogeneity not visible in bulk analyses.

• Spatial Transcriptomics: Maintains spatial information while profiling gene expression, providing insight into ankrd46 expression in tissue contexts.

• CyTOF and Single-Cell Proteomics: Allows simultaneous measurement of multiple proteins at single-cell resolution, capturing post-transcriptional regulation.

Functional Genomics and Systems Biology:

• ChIP-Seq and CUT&RUN: Identifies genomic binding sites of transcription factors that regulate ankrd46 expression.

• ATAC-Seq: Maps chromatin accessibility around the ankrd46 locus during development and in response to stimuli.

• Ribosome Profiling: Measures translation efficiency of ankrd46 mRNA, revealing post-transcriptional regulation.

Protein Engineering and Biochemistry:

• Proximity Labeling: BioID or APEX2 fusions with ankrd46 enable in vivo identification of protein interaction networks.

• Split Fluorescent Proteins: Techniques like BiFC allow visualization of specific protein-protein interactions in live zebrafish.

• Optogenetics: Light-controlled activation or inhibition of ankrd46 function with spatial and temporal precision.

Physiological and Behavioral Analysis:

• Automated Swimming Analysis: High-throughput systems for quantifying swim patterns and responses to stimuli in ankrd46-modified fish.

• Microfluidic Chambers: Allow precise control of environmental conditions while imaging or measuring physiological parameters.

• Non-invasive Cardiac Imaging: Techniques like optical coherence tomography for detailed assessment of cardiac function in ankrd46 mutants.

Integration of these technologies within a zebrafish model system would enable unprecedented insights into ankrd46 function, from molecular interactions to organismal phenotypes, significantly advancing our understanding of this protein's role in development, physiology, and disease.