Recombinant Danio rerio Immediate early response 3-interacting protein 1 (ier3ip1)

What is the basic structure of Danio rerio ier3ip1?

Danio rerio ier3ip1 is the zebrafish ortholog of the human IER3IP1 protein. Based on comparative analysis with human IER3IP1, zebrafish ier3ip1 is likely a small endoplasmic reticulum (ER) membrane protein of approximately 82 amino acids. The protein contains a G-patch domain, which is commonly found in RNA-associated proteins and is suggested to be involved in RNA binding . Like its human counterpart, zebrafish ier3ip1 is expected to have a transmembrane domain at its C-terminal region that anchors it to the ER membrane . The compact structure of this protein makes it particularly interesting for studying fundamental protein-protein interactions in the secretory pathway.

What is the primary cellular localization of ier3ip1 in zebrafish cells?

Based on studies of human IER3IP1, zebrafish ier3ip1 is most likely localized to the endoplasmic reticulum (ER) and potentially enriched in the perinuclear region . The protein may partially overlap with ER exit sites (ERES) and the ER-Golgi intermediate compartment (ERGIC) . Immunofluorescence studies of human IER3IP1 have shown that it cycles between the ER, ERGIC, and the Golgi apparatus . In experimental settings, researchers can confirm the subcellular localization of recombinant zebrafish ier3ip1 through co-localization studies with established ER markers, similar to the methods used for human IER3IP1 .

What is the expression pattern of ier3ip1 during zebrafish development?

While the search results don't provide zebrafish-specific expression data, we can infer from human studies that ier3ip1 is likely expressed in multiple tissues during development. In humans, IER3IP1 shows high expression in heart, skeletal muscle, and kidney, moderate expression in liver and brain, and low expression in placenta, lung, and peripheral blood leukocytes . In zebrafish embryos, researchers would typically analyze ier3ip1 expression through methods such as whole-mount in situ hybridization or qPCR at different developmental stages. The expression pattern during neural development would be particularly relevant given the established role of IER3IP1 in neuronal migration and positioning in mammalian systems .

What are the optimal conditions for expressing recombinant Danio rerio ier3ip1 in bacterial systems?

For bacterial expression of recombinant zebrafish ier3ip1, researchers should consider the following methodological approach:

• Vector selection: Due to the small size of ier3ip1 (approximately 82 amino acids), fusion tags like 6×His, GST, or MBP can facilitate purification while enhancing solubility.

• Expression strain: BL21(DE3) or Rosetta(DE3) E. coli strains are recommended, with the latter being preferable if the zebrafish protein contains rare codons.

• Induction conditions: Lower temperatures (16-20°C) after induction with 0.1-0.5 mM IPTG often yield better results for membrane proteins.

• Solubilization: Since ier3ip1 contains a transmembrane domain, solubilization with mild detergents such as n-dodecyl-β-D-maltoside (DDM) or CHAPS is typically necessary.

• Purification strategy: Two-step purification using affinity chromatography followed by size exclusion chromatography often produces the highest purity.

It's worth noting that due to its transmembrane domain, expressing the full-length protein in bacterial systems might be challenging. Therefore, researchers sometimes opt to express only the soluble domains for functional studies.

What techniques are most effective for studying ier3ip1 interactions with cargo proteins?

Based on approaches used with human IER3IP1, several techniques can be employed to study zebrafish ier3ip1 interactions:

• Co-immunoprecipitation (Co-IP): This can identify proteins that physically interact with ier3ip1 in zebrafish cell lysates. For recombinant studies, tagged ier3ip1 can be used as bait to pull down interacting partners.

• Proximity-based labeling: BioID or APEX2 fusions to ier3ip1 can identify proximal proteins in the native cellular environment.

• Secretome and cell-surface proteomics: As demonstrated with human IER3IP1, comparison of secreted and surface proteins between wild-type and ier3ip1-deficient cells can reveal cargo dependencies .

• FRET or BRET assays: These can measure direct interactions between ier3ip1 and suspected cargo proteins in living cells.

• Yeast two-hybrid screening: Though this approach has limitations for membrane proteins, modified membrane yeast two-hybrid systems can be employed.

When designing these experiments, researchers should consider that IER3IP1 has been shown to interact with ERGIC53 and influence the trafficking of specific proteins including SERPINA1 in human studies .

How can I generate and validate zebrafish models with ier3ip1 mutations?

To generate zebrafish models with ier3ip1 mutations, researchers can employ the following methodological workflow:

• CRISPR/Cas9 genome editing: Design sgRNAs targeting conserved regions of zebrafish ier3ip1, particularly those corresponding to known pathogenic mutations in humans (e.g., L78P or V21G equivalents) .

• Morpholino knockdown: As a complementary or alternative approach, antisense morpholinos can be used for transient knockdown, similar to the in-utero electroporation approach used for mouse Ier3ip1 knockdown .

• Validation strategies:

  • Molecular validation: Confirm mutations by sequencing and protein expression analysis

  • Phenotypic validation: Assess for microcephaly, neuronal migration defects, and other developmental abnormalities

  • Functional validation: Examine ER-Golgi transport using trafficking assays with known cargo proteins

• Rescue experiments: Test whether human IER3IP1 can rescue zebrafish phenotypes to demonstrate functional conservation.

• Live imaging: Exploit the transparency of zebrafish embryos to perform live imaging of neuronal migration and ER-Golgi trafficking in ier3ip1 mutants.

This approach would allow for detailed investigation of ier3ip1 function in a vertebrate model system with excellent optical properties for developmental studies.

How does zebrafish ier3ip1 function in the early secretory pathway?

Based on studies of human IER3IP1, zebrafish ier3ip1 likely functions in the early secretory pathway as follows:

• Component of ER export: Zebrafish ier3ip1 is expected to localize to ER exit sites (ERES) and participate in the selective export of specific cargo proteins from the ER to the Golgi apparatus .

• Cargo selectivity: Like its human counterpart, zebrafish ier3ip1 may control the export of a subgroup of proteins from the ER, suggesting a role in selective recruitment to ERES .

• Receptor trafficking: It may influence the trafficking of cargo receptors such as ERGIC53 and KDEL-receptor 2, as observed in human cells .

• Prevention of inappropriate protein secretion: One function appears to be preventing the inappropriate secretion of ER-resident proteins such as BiP, calreticulin, and calnexin .

• Cycling between compartments: The protein likely cycles between the ER, ERGIC, and Golgi, as demonstrated by studies showing enhanced colocalization with Sec31A when ER export is blocked by a GTP-locked Sar1a mutant .

Understanding these functions in zebrafish could provide valuable insights into conserved mechanisms of early secretory pathway regulation across vertebrates.

What specific cargo proteins depend on ier3ip1 for proper trafficking in zebrafish?

While specific zebrafish cargo proteins haven't been directly identified in the search results, we can extrapolate from human studies that zebrafish ier3ip1 likely regulates the trafficking of proteins involved in:

• Neuronal migration and axon guidance: Potential cargoes may include receptors like UNC5B (netrin receptor), FGFRs, and semaphorin receptors like neuropilin 1, based on the human data .

• Extracellular matrix interactions: This could include integrins (ITGA3, ITGA5, ITGB1) and laminin subunits, which showed altered surface expression in human IER3IP1-deficient cells .

• Growth factor signaling: Receptors such as FGFR2, FGFR3, MET, and components of the TGF-beta pathway (TGFBR3, ACVR2B) might be regulated by zebrafish ier3ip1 .

• Secreted factors: BMP family members and SERPINA1 (alpha-1-antitrypsin) have been identified as affected by human IER3IP1 deficiency .

To identify zebrafish-specific cargo proteins, researchers would need to perform comparative proteomics of secreted and surface proteins in wild-type versus ier3ip1-deficient zebrafish cells, similar to the approach used in human cells.

How do mutations in zebrafish ier3ip1 affect neuronal development?

Based on studies of IER3IP1 in mammalian systems, mutations in zebrafish ier3ip1 would likely affect neuronal development in several ways:

• Altered neuronal morphology: In mouse models, Ier3ip1 knockdown led to neurons with longer neurites in the intermediate zone and more complex arborizations in the cortical plate .

• Disrupted neuronal orientation: Neurons lacking Ier3ip1 showed greater variability in the angles of their leading neurites relative to the cortical plate orientation .

• Changes in cell surface receptor composition: Altered trafficking of guidance receptors could disrupt the neuron's ability to respond appropriately to extracellular signals critical for migration .

• Impaired neuronal migration: The combined effects on morphology and receptor trafficking would likely lead to defects in neuronal positioning, potentially contributing to microcephaly and simplified gyral patterns as seen in human MEDS1 syndrome .

• Disturbances in neurite extension/retraction dynamics: Ier3ip1 deficiency appears to affect the dynamic transition between morphological stages during neuronal differentiation .

These effects would need to be validated in zebrafish models using techniques such as in vivo imaging of fluorescently labeled neurons during brain development.

Can zebrafish ier3ip1 mutants serve as models for human MEDS1 syndrome?

Zebrafish ier3ip1 mutants have significant potential as models for human MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), though with some important considerations:

Creating a panel of different mutants corresponding to human pathogenic variants would provide valuable insights into genotype-phenotype correlations in MEDS1 syndrome.

How can high-throughput screening be designed to identify compounds that rescue ier3ip1 mutant phenotypes?

A comprehensive high-throughput screening approach to identify compounds that rescue ier3ip1 mutant phenotypes could include:

This approach leverages the advantages of the zebrafish system—rapid development, optical clarity, and ease of compound delivery—to efficiently identify potential therapeutic compounds for further development.

What are the challenges and solutions for structural studies of recombinant zebrafish ier3ip1?

Structural studies of recombinant zebrafish ier3ip1 present several challenges due to its properties as a small membrane protein:

• Challenges in protein expression and purification:

  • Membrane integration: The C-terminal transmembrane domain complicates expression in bacterial systems

  • Small size: At approximately 82 amino acids, the protein may be difficult to work with in isolation

  • Potential conformational flexibility: If ier3ip1 cycles between different compartments, it may adopt multiple conformations

• Technical solutions:

  • Expression strategies:

    • Fusion constructs with crystallization chaperones like T4 lysozyme

    • Cell-free expression systems for membrane proteins

    • Insect cell expression with optimized secretion signals

  • Purification approaches:

    • Styrene maleic acid lipid particles (SMALPs) to maintain native lipid environment

    • Amphipols or nanodiscs to stabilize the membrane domain

    • Fragment-based approaches focusing on soluble domains

• Structural determination methods:

  • X-ray crystallography: Challenging but possible with appropriate crystallization chaperones

  • Cryo-EM: Single particle analysis may be difficult due to small size, but could be attempted with larger binding partners

  • NMR spectroscopy: Suitable for smaller proteins, can provide dynamic information

  • Cross-linking mass spectrometry: To map interaction interfaces with binding partners

• Computational approaches:

  • Molecular dynamics simulations to study membrane integration

  • AlphaFold2 or RoseTTAFold predictions as starting models

  • Integrative structural biology combining multiple experimental data sources

Understanding the structure of ier3ip1 would provide critical insights into how this small protein can selectively facilitate the export of specific cargo proteins from the ER.

How does the function of ier3ip1 compare between zebrafish and mammalian models?

Comparative analysis of ier3ip1 function between zebrafish and mammalian models provides valuable evolutionary insights:

• Structural conservation:

  • Sequence homology: Assessment of sequence conservation, particularly in functional domains like the G-patch and transmembrane regions

  • Predicted structural features: Comparison of secondary structure elements and potential interaction interfaces

• Functional conservation:

  • ER-Golgi trafficking: Determine whether zebrafish ier3ip1 controls the export of orthologous cargo proteins identified in mammalian studies

  • Interaction partners: Compare binding affinities with conserved partners such as ERGIC53 and KDEL receptors

  • Rescue experiments: Test whether zebrafish ier3ip1 can complement human IER3IP1 deficiency in cellular models and vice versa

• Developmental roles:

  • Neuronal migration: Compare effects on neuronal positioning and morphology between species

  • Tissue-specific expression: Assess whether expression patterns in developing tissues are conserved

  • Phenotypic severity: Compare the consequences of complete knockout between zebrafish and mouse models

• Methodological table for cross-species comparison:

• Species-specific adaptations:

  • Identify potential differences in cargo repertoire reflecting ecological adaptations

  • Assess compensation mechanisms that may differ between zebrafish and mammals

This comparative approach not only validates zebrafish as a model for human disease but also provides evolutionary context for the conserved functions of this important trafficking regulator.

What are the most promising future research directions for zebrafish ier3ip1?

The study of zebrafish ier3ip1 offers several promising research directions:

• Developmental neurobiology: Leveraging the transparency of zebrafish embryos to perform live imaging of neuronal migration and morphogenesis in ier3ip1 mutants could provide unprecedented insights into how trafficking defects manifest during brain development.

• Drug discovery: The zebrafish model is ideally suited for small molecule screening to identify compounds that rescue ier3ip1-related defects, potentially leading to therapeutic approaches for MEDS1 syndrome.

• Tissue-specific functions: Investigation of ier3ip1 roles beyond the nervous system, particularly in pancreatic development (relevant to the diabetes aspect of MEDS1) and cardiac tissue where human IER3IP1 shows high expression .

• Interaction network mapping: Comprehensive identification of zebrafish ier3ip1 interacting partners across different developmental stages could reveal context-specific functions.

• Evolutionary biology: Comparative analysis of ier3ip1 function across vertebrate species could illuminate how this small protein has evolved to regulate specific aspects of the secretory pathway.