Recombinant Danio rerio Calcium-binding and coiled-coil domain-containing protein 1 (calcoco1), partial

What is the molecular structure of Danio rerio CALCOCO1?

Danio rerio CALCOCO1 shares structural similarity with its mammalian ortholog, featuring an N-terminal SKIP carboxyl homology (SKICH) domain, middle coiled-coil regions (CC), and a carboxy terminal (CT) domain. The protein contains three distinct coiled-coil regions (CC1-3) that are critical for its function and self-interaction. Like its paralogs TAX1BP1 and NDP52, CALCOCO1 likely contains an atypical LC3-interacting region (LIR) motif in the linker region between the SKICH domain and coiled-coil domain . For precise structural characterization, researchers should employ bioinformatic analysis comparing zebrafish CALCOCO1 with mammalian orthologs, followed by experimental validation through domain mapping and mutagenesis studies.

How does CALCOCO1 function in autophagy pathways?

CALCOCO1 functions as an ER-phagy receptor, facilitating the selective degradation of tubular endoplasmic reticulum during proteotoxic and nutrient stress. In mammalian systems, CALCOCO1 binds directly to ATG8 proteins through both LIR and UDS-interacting region (UIR) motifs that function cooperatively . It interacts with VAMP-associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT-like motif, which is essential for its ER-phagy function . Unlike other ER-phagy receptors, CALCOCO1 is not an integral ER membrane protein but instead associates peripherally with the ER, functioning as a soluble ER-phagy receptor . In zebrafish models, researchers should investigate whether these mechanisms are conserved and how they might be adapted to fish-specific physiological contexts.

What experimental evidence supports CALCOCO1's role in selective autophagy?

Several lines of experimental evidence establish CALCOCO1's role in selective autophagy. Studies show that CALCOCO1 depletion causes expansion of the ER and inefficient basal autophagy flux . When CALCOCO1 is absent, cells show higher basal levels of LC3B-II, GABARAP-II, and selective autophagy receptors including p62, NBR1, and NDP52, suggesting impaired degradation . Reconstitution experiments demonstrate that introducing CALCOCO1 back into knockout cells rescues these defects . Additionally, CALCOCO1 co-localizes with early autophagy markers WIPI2 and ATG13 in cytoplasmic puncta during starvation, indicating its recruitment to early autophagic structures . Specifically for ER-phagy, CALCOCO1 knockout impairs the autophagic degradation of tubular ER proteins like VAPA during starvation .

What are optimal conditions for recombinant expression of Danio rerio CALCOCO1?

For successful recombinant expression of Danio rerio CALCOCO1, consider the following optimized protocol:

• Expression system selection: While E. coli systems (BL21(DE3) or Rosetta strains) offer high yield potential, the complex domain structure of CALCOCO1 often benefits from eukaryotic expression systems such as insect cells (Sf9) or mammalian cells (HEK293) that provide appropriate post-translational modifications and folding machinery.

• Vector design: Include purification tags such as His6 or GST at either the N-terminus or C-terminus. For zebrafish proteins, vectors like pIRES2EGFP and pCI-neo have been successfully used in previous studies . Consider codon optimization for your chosen expression system.

• Expression conditions: For bacterial expression, induce at lower temperatures (16-18°C) to improve folding. For mammalian expression, transfect at 70-80% confluency and harvest 48-72 hours post-transfection.

• Solubility considerations: The coiled-coil domains in CALCOCO1 can promote aggregation. Include solubility-enhancing tags like MBP or SUMO, or consider expressing individual domains separately if the full-length protein proves problematic.

What purification strategies yield the highest purity and activity for recombinant CALCOCO1?

A multi-step purification strategy is recommended for obtaining high-purity, active CALCOCO1:

• Initial capture: Affinity chromatography using the fusion tag (Ni-NTA for His-tagged protein or glutathione sepharose for GST-tagged protein) with buffers containing 150-300 mM NaCl, 20-50 mM Tris or HEPES (pH 7.5-8.0), and 5-10% glycerol to maintain stability.

• Intermediate purification: Ion exchange chromatography based on the predicted isoelectric point of zebrafish CALCOCO1, which removes contaminants with different charge properties.

• Polishing: Size exclusion chromatography to separate oligomeric states and ensure homogeneity. Since CALCOCO1 forms homomeric complexes through its coiled-coil domains (particularly CC3), careful characterization of oligomeric state is crucial .

• Buffer optimization: Screen various buffer compositions including different pH ranges (7.0-8.0), salt concentrations (150-500 mM NaCl), and stabilizing additives (glycerol, reducing agents like DTT or TCEP, and potentially mild detergents if membrane association is important).

• Quality control: Verify protein identity by mass spectrometry, purity by SDS-PAGE, and proper folding by circular dichroism or thermal shift assays.

How can I verify the functional activity of recombinant Danio rerio CALCOCO1?

To confirm that your recombinant zebrafish CALCOCO1 is functionally active:

• Binding assays: Perform pull-down assays with zebrafish ATG8 proteins (LC3/GABARAP family) to verify LIR/UIR functionality. Also test interaction with VAPA/VAPB to confirm FFAT-mediated binding. Self-interaction assays should demonstrate homomerization capability, particularly through the CC3 domain .

• Structural verification: Use circular dichroism spectroscopy to confirm secondary structure elements, particularly the alpha-helical content expected in coiled-coil regions. Thermal stability assays can assess proper folding.

• Cellular activity: The ultimate functional verification involves rescue experiments in CALCOCO1-knockout cells. Expression of functional recombinant CALCOCO1 should normalize elevated levels of LC3B-II, GABARAP-II, p62, NBR1, and NDP52 observed in knockout cells .

• ER-phagy assays: Test whether the recombinant protein can restore ER turnover during starvation using assays like tandem fluorescent-tagged ER protein degradation or monitoring ER protein levels by Western blot.

What are effective approaches for generating CALCOCO1 knockout models in zebrafish?

For generating CALCOCO1 loss-of-function models in zebrafish:

• CRISPR/Cas9 genome editing: Design multiple sgRNAs targeting early exons of zebrafish calcoco1 to create frameshift mutations. The search results indicate CRISPR/Cas9 has been successfully used to generate CALCOCO1 knockouts in mammalian cells, suggesting this approach should transfer well to zebrafish . Key steps include:

  • Design 2-3 sgRNAs with minimal off-target potential

  • Microinject sgRNA and Cas9 mRNA/protein into single-cell embryos

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

  • Raise F0 fish to adulthood and screen for germline transmission

  • Establish and characterize stable F2 homozygous lines

• Morpholino knockdown: For rapid preliminary studies, design splice-blocking or translation-blocking morpholinos. Critical validation controls include:

  • Rescue experiments with wildtype mRNA to confirm specificity

  • Western blotting to verify protein reduction

  • Comparison with CRISPR mutant phenotypes when available

• Conditional approaches: For studying CALCOCO1 in specific tissues or developmental stages:

  • Use Gal4/UAS or Cre/loxP systems for tissue-specific knockout

  • Consider heat-shock inducible dominant-negative constructs

  • Employ photoactivatable morpholinos for spatiotemporal control

What methods are most effective for monitoring CALCOCO1-mediated ER-phagy in zebrafish?

To effectively monitor CALCOCO1-mediated ER-phagy in zebrafish cells or tissues:

• Autophagic flux assays: Measure ER protein turnover by comparing their levels with and without lysosomal inhibitors like Bafilomycin A1. In mammalian systems, CALCOCO1 specifically mediates degradation of tubular ER proteins such as VAPA, RTN3, and TEX264 during starvation . Western blot analysis should show accumulation of these proteins in CALCOCO1-deficient cells treated with starvation media plus Bafilomycin A1 compared to starvation alone.

• Fluorescence-based methods: Employ dual-color reporters to directly visualize ER-phagy:

  • Express tandem mCherry-EYFP-tagged ER proteins (like VAPA) in wildtype and CALCOCO1-knockout backgrounds

  • During ER-phagy, these constructs appear as red-only puncta after EYFP quenching in acidic autolysosomes

  • Quantify the ratio of red-only puncta to total puncta as a measure of ER-phagy flux

• ER morphology analysis: Assess ER volume and distribution using:

  • Confocal microscopy with ER-specific dyes or antibodies against ER markers

  • Transmission electron microscopy to directly visualize ER structure and autophagosome formation

  • Quantitative analysis of ER protein levels by Western blot

• Co-localization studies: Examine the co-localization of CALCOCO1 with:

  • ER markers (VAPA/B, RTN3, etc.)

  • Early autophagy markers (WIPI2, ATG13) during starvation

  • Autolysosomal markers to track the complete degradation process

How can contradictory results in CALCOCO1 functional assays be reconciled?

When facing contradictory results in CALCOCO1 studies, employ these systematic approaches:

• Context-dependent function analysis:

  • CALCOCO1 shows important functional distinctions that may explain contradictory results. For instance, mammalian CALCOCO1 promotes basal autophagy but is not required for starvation-induced bulk autophagy . Similarly, it specifically targets tubular ER (affecting proteins like VAPA) but not sheet ER (leaving FAM134B degradation unaffected) .

  • Test different cellular conditions systematically, comparing basal versus stressed states

  • Examine tissue-specific effects that might explain discrepancies

• Technical considerations:

  • Verify complete knockout versus partial knockdown effects

  • Assess potential compensation by paralogs (TAX1BP1, NDP52)

  • Evaluate tag interference with function when using fusion proteins

  • Confirm antibody specificity with appropriate knockout controls

• Experimental harmonization:

  • Standardize starvation protocols (duration, media composition)

  • Use multiple complementary assays (e.g., both Western blot and fluorescent reporter methods)

  • Compare identical timepoints when evaluating dynamic processes

  • Employ both gain-of-function and loss-of-function approaches

• Quantitative analysis:

  • Perform rigorous statistical analysis across multiple independent experiments

  • Consider threshold effects or non-linear responses

  • Report effect sizes along with statistical significance

How does stress affect CALCOCO1 function in zebrafish models?

Stress conditions significantly influence CALCOCO1 function in cellular models, with likely similar effects in zebrafish:

• Nutrient stress response:

  • During starvation, CALCOCO1 facilitates the selective degradation of excess ER components to restore homeostasis

  • The number of CALCOCO1-positive cytoplasmic puncta increases during starvation conditions

  • In HBSS-treated cells, CALCOCO1 promotes the degradation of tubular ER proteins including RTN3, TEX264, VAPA, and VAPB

• Proteotoxic stress response:

  • CALCOCO1 acts as a receptor for degradation of tubular ER in response to both nutrient and proteotoxic stress

  • This function helps prevent ER expansion that would otherwise occur during stress conditions

  • Depletion of CALCOCO1 leads to expansion of the ER upon nutrient stress

• Stress-induced regulation:

  • CALCOCO1 itself is degraded by macro-autophagy

  • It relocates to early autophagic structures during stress, co-localizing with WIPI2 and ATG13

  • Under basal conditions, CALCOCO1 promotes efficient autophagy flux, but this role appears distinct from its function during starvation-induced bulk autophagy

To study these stress responses in zebrafish models, researchers should examine CALCOCO1 localization, ER morphology, and autophagy markers under controlled stress conditions at both cellular and organismal levels.

What is the potential for CALCOCO1 as a therapeutic target based on zebrafish studies?

Zebrafish models provide valuable insights into CALCOCO1's therapeutic potential:

• Modulating ER homeostasis:

  • Since CALCOCO1 regulates ER morphology and turnover during stress , compounds that modulate its activity could potentially influence ER homeostasis in diseases characterized by ER dysfunction

  • Zebrafish offer a vertebrate model for medium-throughput screening of such compounds

• Targeting selective autophagy:

  • CALCOCO1's role in basal autophagy suggests therapeutic applications in conditions with autophagy dysfunction

  • The ability to visualize autophagy processes in transparent zebrafish embryos provides advantages for compound screening

  • Zebrafish could help identify autophagy modulators with specificity for CALCOCO1-mediated pathways

• Neurological and metabolic applications:

  • Since ER stress and autophagy dysfunction are implicated in various neurological and metabolic diseases, CALCOCO1 modulators may have therapeutic potential in these areas

  • Zebrafish models of such diseases could evaluate whether CALCOCO1 modulation affects disease progression

• Developmental and regenerative medicine:

  • Understanding CALCOCO1's role in zebrafish development and tissue regeneration could reveal therapeutic applications in regenerative medicine

  • Zebrafish heart and fin regeneration models could assess whether CALCOCO1 modulation enhances repair processes

Before pursuing therapeutic development, researchers should thoroughly characterize CALCOCO1 function in zebrafish and validate findings in mammalian models to ensure translational relevance.

How do CALCOCO1 interactomes differ between zebrafish and mammals?

Understanding interactome differences between zebrafish and mammalian CALCOCO1 requires comprehensive comparative studies:

• Core conserved interactions:

  • Mammalian CALCOCO1 interacts with ATG8 proteins via LIR and UIR motifs

  • It binds VAMP-associated proteins VAPA and VAPB through a FFAT-like motif

  • Self-interaction occurs primarily through the CC3 domain

  • These core interactions are likely conserved in zebrafish CALCOCO1, though binding affinities may differ

• Species-specific interactions:

  • Zebrafish-specific binding partners may exist due to evolutionary divergence

  • Differences in protein isoforms between species could affect interaction profiles

  • Tissue-specific interactors may vary based on the expression patterns in each species

• Methodology for comparative interactomics:

  • Perform parallel affinity purification-mass spectrometry (AP-MS) in zebrafish and mammalian systems

  • Express both zebrafish and mammalian CALCOCO1 in each system to control for cellular context

  • Compare interactomes under both basal and stress conditions

  • Validate key interactions with direct binding assays and functional studies

• Functional implications:

  • Differences in interactomes could explain species-specific functions or regulations

  • Conservation of key interactions would support the use of zebrafish as a model for human CALCOCO1 function

  • Understanding these differences is crucial for translating findings between model systems

What role might CALCOCO1 play in zebrafish development and organ function?

While the search results don't provide direct information on CALCOCO1's developmental roles, several hypotheses can be formulated based on its cellular functions:

• ER remodeling during development:

  • The ER undergoes significant remodeling during cell differentiation and organ development

  • CALCOCO1's role in ER-phagy suggests it may contribute to developmental ER remodeling

  • Key developmental processes requiring extensive protein synthesis and secretion (like notochord development or neuron differentiation) might particularly depend on CALCOCO1

• Stress adaptation during organogenesis:

  • Developing organs experience various stresses, including nutrient fluctuations

  • CALCOCO1's role in stress adaptation through ER-phagy might be crucial during organ formation

  • This function could be particularly important in highly secretory organs like the pancreas, liver, or hatching gland

• Potential tissue-specific functions:

  • Expression analysis across developmental stages and tissues would reveal where CALCOCO1 might have prominent roles

  • Given its role in ER homeostasis , tissues with extensive ER networks might particularly depend on CALCOCO1

  • The function might differ between tissues based on their specific autophagy requirements

• Developmental timing of CALCOCO1 requirement:

  • CALCOCO1 knockout phenotypes might manifest at specific developmental stages coinciding with increased autophagy demand

  • Early development relies heavily on maternal contribution, so zygotic knockout effects might only become apparent at later stages

  • Temporal control of CALCOCO1 function using conditional approaches would help delineate stage-specific requirements

What are the most promising areas for future research on Danio rerio CALCOCO1?

Several high-priority research directions for zebrafish CALCOCO1 include:

• Comparative biology:

  • Detailed characterization of zebrafish CALCOCO1 structure compared to mammalian orthologs

  • Analysis of functional conservation and divergence across vertebrates

  • Investigation of whether gene duplication events in teleost fish have resulted in subfunctionalization of CALCOCO1 paralogs

• Developmental roles:

  • Temporal and spatial expression profiling throughout zebrafish development

  • Phenotypic characterization of CALCOCO1 mutants during embryogenesis and larval development

  • Investigation of tissue-specific requirements using conditional approaches

• Stress response mechanisms:

  • Characterization of how different stressors affect CALCOCO1 function in zebrafish

  • Live imaging of CALCOCO1-mediated ER-phagy during stress responses

  • Comparative analysis of stress tolerance between wildtype and CALCOCO1-deficient zebrafish

• Interactome mapping:

  • Comprehensive identification of zebrafish CALCOCO1 binding partners

  • Comparison with mammalian interactomes to identify conserved and divergent interactions

  • Structure-function analysis of key protein-protein interactions

• Disease modeling:

  • Generation of zebrafish models with human disease-associated CALCOCO1 variants

  • Investigation of CALCOCO1's role in zebrafish models of neurodegenerative diseases

  • Screening for compounds that modulate CALCOCO1-dependent processes

What technological advances would facilitate research on zebrafish CALCOCO1?

Several technological developments would significantly advance zebrafish CALCOCO1 research:

• Genome editing improvements:

  • Base editing or prime editing methods for precise introduction of specific mutations

  • Enhanced homology-directed repair efficiency for knock-in generation

  • Multiplex CRISPR approaches for simultaneous modification of CALCOCO1 and interacting partners

• Imaging advancements:

  • Improved live imaging techniques for visualizing ER-phagy in intact zebrafish

  • Super-resolution microscopy adapted for zebrafish tissues

  • Correlative light and electron microscopy for connecting CALCOCO1 localization with ultrastructural features

• Protein analysis tools:

  • Zebrafish-specific antibodies against CALCOCO1 and related proteins

  • Improved methods for isolation of subcellular compartments from zebrafish tissues

  • Adapted proximity labeling techniques for in vivo interactome mapping

• Single-cell approaches:

  • Single-cell proteomics from zebrafish tissues to analyze CALCOCO1 levels and modifications

  • Single-cell transcriptomics to identify pathways affected by CALCOCO1 deficiency

  • Spatial transcriptomics to map expression patterns with higher resolution

• Drug screening platforms:

  • High-content screening methods optimized for zebrafish CALCOCO1-related phenotypes

  • Microfluidic systems for automated analysis of zebrafish responses to compounds

  • In silico modeling of CALCOCO1 for virtual screening of potential modulators