Recombinant Danio rerio Tetratricopeptide repeat protein 39A (ttc39a), partial

What is the basic structure of Danio rerio TTC39A protein?

The zebrafish (Danio rerio) TTC39A protein consists of 598 amino acids and shares structural similarities with its human ortholog. The primary feature of TTC39A is the domain of unknown function 3808 (DUF3808), which spans almost the entire protein . This domain is generally considered to be associated with outer mitochondrial membrane proteins and has been conserved from fungi to humans . The protein is predicted to contain multiple alpha helices (similar to the 12 helices in the human version) and likely contains tetratricopeptide repeats and a transmembrane domain .

What is the evolutionary conservation of TTC39A between zebrafish and humans?

The zebrafish TTC39A ortholog shows considerable conservation with human TTC39A, demonstrating 60.8% protein identity and 76.0% protein similarity . This high degree of conservation suggests potential functional similarity and makes zebrafish an appropriate model organism for studying TTC39A functions. The table below illustrates protein conservation across species:

What is known about the expression pattern of TTC39A in zebrafish?

While the search results don't specifically detail the expression pattern of TTC39A in zebrafish, we can infer from human data that TTC39A may be expressed in multiple tissues . In humans, TTC39A shows high expression in mammary glands and testis, with lower expression in the immune system . Expression patterns in zebrafish would need to be confirmed through experimental techniques such as RT-PCR or in situ hybridization. The RT-PCR approaches used for confirming hox gene expression in zebrafish could be adapted for studying TTC39A expression .

What are the key considerations when designing experiments with recombinant Danio rerio TTC39A?

When working with recombinant Danio rerio TTC39A, researchers should consider several critical factors that influence experimental outcomes:

• Protein expression and purification: TTC39A contains a predicted transmembrane domain, which may complicate bacterial expression systems. Consider using eukaryotic expression systems or adding solubility tags.

• Zebrafish strain selection: Different laboratories use different zebrafish strains including AB wild-type, AB Tg, or 5D Tropical, which can influence experimental outcomes . For consistency with published data, researchers should consider strain selection carefully.

• Experimental conditions: Parameters such as temperature, pH, and buffer composition can affect protein stability and function.

• Positive and negative controls: Include appropriate controls, especially given the relatively unknown function of TTC39A.

• Validation techniques: Multiple approaches (Western blotting, mass spectrometry) should be used to confirm protein identity and integrity.

How should researchers optimize expression and purification of recombinant zebrafish TTC39A?

Optimizing expression and purification of recombinant zebrafish TTC39A requires addressing several challenges:

• Expression system selection: E. coli systems may be inadequate for proper folding of complex proteins like TTC39A. Consider baculovirus-insect cell or mammalian expression systems for proper post-translational modifications.

• Construct design: Since full-length TTC39A contains a transmembrane domain, consider expressing soluble domains separately or using solubility-enhancing tags (MBP, SUMO, or GST).

• Purification strategy: Implement a multi-step purification protocol:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Intermediate purification using ion-exchange chromatography

  • Polishing step using size-exclusion chromatography

• Protein stability: Include appropriate stabilizers in buffer systems based on thermal shift assays to identify optimal conditions.

• Functional validation: Develop activity assays to ensure the purified protein maintains native functionality.

What chorion considerations are important when studying TTC39A in zebrafish embryos?

The chorion status (intact versus removed) is a critical parameter in zebrafish experiments that can significantly impact experimental outcomes . When studying TTC39A in zebrafish embryos:

• Dechorionation effects: Removing the chorion increases permeability to compounds and may alter developmental processes. In the DNT-DIVER database comparisons, laboratories differed in their dechorionation protocols, with Labs B and C performing dechorionation while Lab A did not .

• Timing considerations: If studying early development, consider that the chorion naturally serves as a barrier. Early removal may affect natural developmental processes.

• Protocol standardization: Based on the findings from zebrafish toxicity studies, dechorionation status should be explicitly reported and standardized across experiments to ensure reproducibility .

• Exposure conditions: The absence of chorion changes exposure dynamics, requiring adjustments to concentration and duration parameters.

How can CRISPR-Cas9 be used for studying TTC39A function in zebrafish?

CRISPR-Cas9 offers powerful approaches for investigating TTC39A function in zebrafish:

• Knockout strategies:

  • Design sgRNAs targeting early exons of ttc39a gene

  • Validate mutations by sequencing and protein expression analysis

  • Analyze phenotypes across multiple developmental stages

  • Implement tissue-specific knockout using tissue-specific promoters

• Knock-in approaches:

  • Insert fluorescent reporters to monitor TTC39A expression patterns

  • Create tagged versions for protein localization studies

  • Introduce specific mutations corresponding to human variants

  • Generate conditional alleles using loxP/Cre systems

• Validation considerations:

  • Screen multiple founder lines to avoid off-target effects

  • Perform complementation tests with morpholinos or other genetic approaches

  • Rescue experiments with wild-type mRNA to confirm specificity

• Phenotypic analysis:

  • Conduct comprehensive developmental analysis

  • Examine tissue-specific functions

  • Perform transcriptomic analysis to identify affected pathways

How can researchers address data variability when studying TTC39A in zebrafish across different laboratories?

The zebrafish community has recognized significant variability in experimental outcomes across laboratories . To address this when studying TTC39A:

• Protocol standardization:

  • Explicitly report all experimental parameters

  • Adopt standardized protocols, particularly for critical factors like exposure conditions and chorion status

  • Implement positive controls with known outcomes

• Quantitative benchmarking:

  • Use benchmark concentration (BMC) modeling approaches to standardize comparisons

  • Report potency values rather than binary outcomes

  • Calculate concordance metrics when comparing across laboratories

• Key parameters to report:

  • Fish strain (e.g., AB wild-type vs. 5D Tropical)

  • Dechorionation status

  • Exposure volume

  • Exposure scenario (static vs. static renewal)

  • Measurement timing

• Replicate studies:

  • Perform experiments in multiple batches

  • Consider inter-laboratory validation for critical findings

What is the potential role of TTC39A in zebrafish development and disease models?

While the specific function of TTC39A remains poorly understood, several lines of investigation could be pursued:

• Developmental role:

  • Temporal expression analysis during embryonic development

  • Spatial mapping using in situ hybridization, similar to approaches used for hox genes

  • Loss-of-function studies examining developmental phenotypes

  • Gain-of-function studies to assess overexpression effects

• Disease modeling:

  • Since human TTC39A is induced by TFAP2C in hormone-responsive breast carcinoma cells , investigate potential roles in hormone-responsive cancer models in zebrafish

  • Explore potential metabolic functions, given the proximity to mitochondrial membranes

  • Investigate interaction with signaling pathways using small molecule modulators

• Interaction studies:

  • Identify protein binding partners through co-immunoprecipitation

  • Map genetic interactions through modifier screens

  • Assess subcellular localization in different tissues and developmental stages

What are the best approaches for studying TTC39A expression patterns in zebrafish?

To comprehensively characterize TTC39A expression in zebrafish:

• Transcriptional analysis:

  • RT-PCR using validated primers spanning exon-exon junctions

  • qPCR for quantitative assessment across tissues and developmental stages

  • RNA-seq for genome-wide expression context

  • Single-cell RNA-seq to identify cell populations expressing TTC39A

• Protein detection:

  • Develop specific antibodies against zebrafish TTC39A

  • Use epitope tagging approaches (if antibodies unavailable)

  • Western blotting for quantitative tissue analysis

  • Immunohistochemistry for spatial localization

• In vivo visualization:

  • Generate transgenic reporter lines (ttc39a:GFP)

  • Perform in situ hybridization for embryonic and larval stages

  • Use time-lapse imaging to track expression dynamics

• Comparative approaches:

  • Analyze expression across multiple zebrafish strains

  • Compare with mammalian expression patterns

How can researchers investigate potential post-translational modifications of zebrafish TTC39A?

Investigating post-translational modifications (PTMs) of zebrafish TTC39A requires specialized approaches:

• Prediction and computational analysis:

  • Use bioinformatic tools to predict likely PTM sites

  • Compare with known or predicted sites in human TTC39A

  • Human TTC39A has several predicted phosphorylation sites and a sulfination site at position 175

• Experimental identification:

  • Mass spectrometry-based proteomics to identify modifications

  • Enrichment strategies for specific modifications (phospho-enrichment, etc.)

  • Site-directed mutagenesis to confirm functional significance

• Functional analysis:

  • Generate antibodies specific to modified forms

  • Create phosphomimetic and phospho-dead mutants

  • Assess impact of mutations on protein localization and function

• Developmental context:

  • Analyze modifications across developmental stages

  • Examine tissue-specific modification patterns

  • Investigate stimulus-dependent modification changes

What techniques are recommended for exploring protein-protein interactions of TTC39A in zebrafish?

To investigate TTC39A interaction partners in zebrafish:

• Affinity purification approaches:

  • Generate tagged TTC39A constructs (FLAG, HA, or BioID)

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Develop stable cell lines or transgenic fish expressing tagged TTC39A

• Proximity labeling methods:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2 peroxidase-based proximity labeling

  • Split-BioID for studying interaction dynamics

• Genetic interaction screens:

  • CRISPR-based screens in zebrafish cells

  • Morpholino or CRISPR combinations to identify genetic interactions

  • Modifier screens in ttc39a mutant backgrounds

• Visualization of interactions:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Proximity ligation assay (PLA) in tissue sections

How do TTC39A paralogs (TTC39B and TTC39C) compare in zebrafish, and what are the implications for functional studies?

Understanding the relationship between TTC39A and its paralogs is crucial for functional studies:

• Evolutionary relationships:

  • TTC39A has two paralogs, TTC39B and TTC39C, likely arising from gene duplication events

  • Compare sequence conservation and domain architecture across paralogs

• Expression pattern comparison:

  • Assess whether paralogs show overlapping or distinct expression patterns

  • Determine whether paralogs might have redundant functions

• Functional redundancy:

  • Design knockout studies that address potential compensation

  • Consider double or triple knockout approaches

  • Assess cross-rescue experiments (can one paralog rescue another's loss?)

• Specialized functions:

  • Investigate whether paralogs have evolved distinct functions

  • Examine paralog-specific protein interaction networks

  • Identify tissues where only specific paralogs are expressed

How does zebrafish TTC39A compare with human TTC39A, and what are the implications for using zebrafish as a model?

Understanding cross-species similarities and differences is critical when using zebrafish as a model:

• Structural comparison:

  • Zebrafish TTC39A (598aa) is slightly shorter than human TTC39A (613aa)

  • The 60.8% protein identity and 76.0% similarity suggest functional conservation

  • Both contain the DUF3808 domain spanning most of the protein

• Expression pattern comparison:

  • Human TTC39A shows high expression in mammary glands and testis

  • Expression mapping in zebrafish would clarify comparative expression patterns

  • Tissue-specific differences may indicate evolved functional specialization

• Model validation:

  • Determine whether phenotypes in zebrafish mutants recapitulate known human conditions

  • Test whether human TTC39A can rescue zebrafish ttc39a mutants

  • Compare protein interaction networks between species

• Translational potential:

  • Assess zebrafish studies for applicability to human biology

  • Consider species-specific differences when interpreting results

What are the common challenges in producing recombinant zebrafish TTC39A protein and how can they be addressed?

Producing recombinant zebrafish TTC39A presents several technical challenges:

• Solubility issues:

  • Challenge: TTC39A contains predicted transmembrane domains that may cause aggregation

  • Solution: Use solubility-enhancing tags (MBP, SUMO), optimize expression conditions, or express soluble domains separately

• Post-translational modifications:

  • Challenge: Bacterial systems lack eukaryotic PTM machinery

  • Solution: Use eukaryotic expression systems (insect cells, mammalian cells) if modifications are critical

• Protein stability:

  • Challenge: Recombinant TTC39A may be prone to degradation

  • Solution: Screen buffer conditions using thermal shift assays, add stabilizers, and optimize purification protocols

• Functional verification:

  • Challenge: Without known function, it's difficult to verify activity

  • Solution: Develop binding assays with predicted partners or structural integrity tests

• Yield limitations:

  • Challenge: Complex proteins often express at low levels

  • Solution: Optimize codon usage, culture conditions, and induction parameters

How can researchers address conflicting data when studying TTC39A in zebrafish?

When confronted with conflicting data regarding zebrafish TTC39A:

• Protocol standardization:

  • Systematically document all experimental variables

  • Implement the quantitative benchmarking approaches used in toxicity studies

  • Report potency values and include measures of variability

• Critical parameter identification:

  • Systematically test key parameters (fish strain, dechorionation status, exposure conditions)

  • Quantify the impact of each parameter on experimental outcomes

  • Create standardized protocols addressing the most influential variables

• Statistical approaches:

  • Use appropriate statistical methods for comparing results across studies

  • Implement meta-analysis techniques when multiple datasets are available

  • Report effect sizes rather than just statistical significance

• Validation strategies:

  • Employ multiple independent techniques to confirm findings

  • Collaborate with other laboratories to validate key findings

  • Consider the benchmark concentration (BMC) modeling approach used in toxicity studies

What are promising future research directions for understanding TTC39A function in zebrafish?

Several promising research directions could advance understanding of TTC39A function:

• Comprehensive expression mapping:

  • Detailed spatial and temporal analysis throughout development

  • Single-cell resolution mapping to identify cell types expressing TTC39A

  • Regulation of expression under various conditions

• Functional genomics:

  • CRISPR-based knockout and knock-in studies

  • Tissue-specific and inducible knockout models

  • Transcriptomic and proteomic analysis of mutants

• Structure-function relationships:

  • Structural characterization of the DUF3808 domain

  • Mutational analysis to identify functional regions

  • Comparative analysis with paralogs and orthologs

• Pathway integration:

  • Identification of upstream regulators and downstream targets

  • Integration with known developmental and physiological pathways

  • Small molecule modulator screening

• Translational studies:

  • Modeling human variants in zebrafish

  • Therapeutic target identification

  • Cross-species validation of findings

How might high-throughput approaches be applied to study TTC39A function in zebrafish?

High-throughput approaches offer powerful tools for studying TTC39A:

• CRISPR screening:

  • Genome-wide CRISPR screens to identify genetic interactions

  • Targeted CRISPR libraries focusing on pathway components

  • Single-cell CRISPR screens to identify cell-specific effects

• Chemical genetics:

  • Small molecule screens to identify modulators of TTC39A function

  • Chemogenomic profiling to place TTC39A in functional networks

  • Structure-based virtual screening for binding partners

• Multi-omics integration:

  • Integrated transcriptomic, proteomic, and metabolomic analysis

  • Network modeling of TTC39A function

  • Machine learning approaches to predict function from omics data

• High-content imaging:

  • Automated phenotyping of TTC39A mutants

  • Real-time imaging of protein dynamics

  • Multiplexed imaging of interaction partners and pathway components

What are the key considerations for researchers beginning work with recombinant Danio rerio TTC39A?

Researchers beginning work with recombinant Danio rerio TTC39A should:

• Acknowledge knowledge gaps: The function of TTC39A remains poorly understood, requiring careful experimental design and multiple approaches.

• Standardize protocols: Pay particular attention to experimental parameters known to affect zebrafish studies, including strain selection, dechorionation status, and exposure conditions .

• Implement comparative approaches: Leverage the evolutionary conservation between zebrafish and human TTC39A (60.8% identity, 76.0% similarity) while acknowledging potential functional differences.

• Develop multiple tools: Generate antibodies, transgenic reporters, and CRISPR mutants to enable comprehensive functional analysis.

• Address technical challenges: Anticipate difficulties with protein expression and purification due to predicted transmembrane domains and develop appropriate strategies.

• Collaborate across disciplines: Combine structural biology, developmental biology, and systems biology approaches to build a comprehensive understanding of TTC39A function.