Recombinant Danio rerio Tumor necrosis factor alpha-induced protein 8-like protein 1 (tnfaip8l1)

What is the protein structure of Danio rerio tnfaip8l1 and how does it compare to other TIPE family members?

Danio rerio tnfaip8l1 is a 186-amino acid protein with the sequence: MDSFSTKNLALQAQKKLMSKMATKTVANLF IDDTSSEVLDELYRVTKEYTRNRKEAQKIIKNLIKMVVKLGVLYRNGQFNNEELALVERFRKKVHTLAMTAVSFYQIDFTFDRRVMSNLLNDCRELLHQAINRHLTAKSHARINHVFNHFADCDFLATLY GPSEVYRGHLQKICEGVNKMLDEGNL .

Like other TIPE family members, it contains a death effector domain (DED). The TIPE family proteins share high structural similarity with approximately 54% homology and 75% amino acid sequence similarities . This conservation suggests functional similarities between zebrafish tnfaip8l1 and its mammalian counterparts. In humans, TIPE1 is expressed in various cell types including hepatocytes, intestinal epithelial cells, muscle tissues, neurons, and germ cells, but is notably absent in mature B and T lymphocytes .

What are the optimal storage conditions for recombinant Danio rerio tnfaip8l1?

Recombinant Danio rerio tnfaip8l1 stability depends on multiple factors, including buffer ingredients and storage temperature. For optimal preservation:

• Lyophilized form: Maintain at -20°C/-80°C for up to 12 months

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Working aliquots: Store at 4°C for no more than one week

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity

For reconstitution, it is recommended to:

• Briefly centrifuge the vial before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL concentration

• Add glycerol to a final concentration of 5-50% (50% is standard)

• Aliquot for long-term storage at -20°C/-80°C

How is tnfaip8l1 expression regulated during inflammatory responses?

Tnfaip8l1 expression is regulated through the TNF-α signaling pathway. The process follows this cascade:

• Immune cells respond to inflammatory stimuli by secreting TNF-α

• TNF-α binds to TNFR1 and TNFR2 receptors

• This activates the NF-κB signaling pathway

• NF-κB activation induces expression of TNFAIP8 family proteins, including tnfaip8l1

Unlike TNFAIP8 which is expressed in most tissues including bone marrow, immune system, gastrointestinal tract, lung, and adipose tissues, TNFAIP8L1 shows more restricted expression patterns. The differential expression suggests tissue-specific functions that must be considered when designing zebrafish experiments as a model for human conditions .

What methodologies are most effective for studying tnfaip8l1 function in zebrafish cancer models?

To effectively study tnfaip8l1 in zebrafish cancer models, researchers should consider these methodological approaches:

Experimental Design Table for Zebrafish tnfaip8l1 Cancer Studies:

Based on studies of TNFAIP8 in clear cell renal cell carcinoma (ccRCC), where it promotes migration and invasion through epithelial-mesenchymal transition (EMT), researchers could apply similar wound healing and transwell assays after overexpression or knockdown of tnfaip8l1 in zebrafish cells . Additionally, monitoring expression of EMT markers would provide insight into whether tnfaip8l1 functions similarly in zebrafish models.

How do the functions of tnfaip8l1 differ between zebrafish and mammalian models in the context of tumor suppression?

The functional comparison between zebrafish tnfaip8l1 and its mammalian counterparts reveals important evolutionary considerations:

In mammalian models, TIPE1 generally functions as a tumor suppressor. It promotes tumor cell apoptosis in several cancers through:

• Interaction with Rac1, promoting tumor cell apoptosis via inhibiting JNK and p65 activity in liver cancer

• Upregulation of pro-apoptotic members of the Bcl-2 family in RAW264.7 cells

To accurately compare functions:

• Conduct parallel knockdown experiments in zebrafish and mammalian cell lines

• Perform rescue experiments with cross-species protein expression

• Analyze downstream signaling pathways to identify conserved and divergent mechanisms

• Consider the evolutionary conservation of interacting partners like Rac1

What are the molecular mechanisms by which tnfaip8l1 regulates apoptosis, and how can these be experimentally verified?

The molecular mechanisms of tnfaip8l1-mediated apoptosis regulation can be experimentally verified through these methodological approaches:

• Protein Interaction Analysis:

  • Co-immunoprecipitation to identify binding partners

  • Proximity ligation assays to confirm protein-protein interactions in situ

  • Yeast two-hybrid screening to discover novel interacting proteins

• Signaling Pathway Analysis:

  • Western blotting to assess phosphorylation status of key signaling molecules (JNK, p65)

  • Analysis of Bcl-2 family protein expression patterns

  • Caspase activity assays to measure apoptotic enzyme activation

• Functional Domain Mapping:

  • Creation of truncation mutants to identify functional domains

  • Site-directed mutagenesis of key residues to disrupt specific interactions

  • Domain swapping with other TIPE family members to assess functional redundancy

Drawing from TNFAIP8 studies, which shows inhibition of caspase-8 activity without affecting procaspase-8 processing, resulting in inhibition of BID cleavage and caspase-3 activation , researchers should assess whether tnfaip8l1 affects similar pathways or employs distinct mechanisms.

How do the expression patterns and functions of all four TNFAIP8 family members compare across species?

The TNFAIP8 family shows distinct expression patterns and functions across different species and tissues:

Comparative Expression and Function of TNFAIP8 Family Proteins:

Methodological approaches for cross-species comparative studies should include:

• Phylogenetic analysis of protein sequences

• Gene expression profiling across tissues in different model organisms

• Cross-species complementation assays to test functional conservation

• Analysis of promoter regions to identify conserved regulatory elements

What experimental approaches are most effective for analyzing tnfaip8l1 involvement in zebrafish inflammatory responses?

To effectively study tnfaip8l1 in zebrafish inflammatory responses, consider these methodological approaches:

• In vivo inflammation models:

  • Lipopolysaccharide (LPS) injection to induce systemic inflammation

  • Tail fin amputation to study tissue regeneration and inflammatory resolution

  • Infection models with bacterial or viral pathogens

  • Chemical-induced inflammation (e.g., copper sulfate for neutrophil recruitment)

• Cellular and molecular analyses:

  • Neutrophil and macrophage migration assays

  • Cytokine expression profiling using qPCR array

  • Phospho-flow cytometry to assess signaling pathway activation

  • RNA-seq of isolated immune cell populations

• Genetic approaches:

  • CRISPR/Cas9 knockout of tnfaip8l1

  • Conditional expression systems (heat shock or tissue-specific)

  • Morpholino knockdown for transient analysis

  • Transgenic reporter lines for real-time inflammation visualization

• Comparative analyses:

  • Parallel experiments with other TIPE family members

  • Rescue experiments with human TIPE1

  • Epistasis studies with NF-κB pathway components

Since mammalian TIPE1 shows distinct expression patterns from TNFAIP8 and is absent in mature lymphocytes, researchers should pay particular attention to cell-type specific expression in zebrafish inflammation models.

What are common challenges in recombinant tnfaip8l1 protein production and application, and how can they be overcome?

Researchers working with recombinant Danio rerio tnfaip8l1 may encounter these common challenges:

Production Challenges and Solutions:

• Low protein yield:

  • Optimize codon usage for expression system

  • Test multiple expression vectors and host strains

  • Adjust induction parameters (temperature, inducer concentration, duration)

  • The current production method uses yeast as the source, which may provide better yields than bacterial systems for this protein

• Protein solubility issues:

  • Modify buffer compositions (pH, salt concentration, additives)

  • Consider fusion tags to enhance solubility (e.g., MBP, SUMO)

  • Test refolding protocols if inclusion bodies form

  • Compare with the >85% purity achieved in current preparations

• Protein stability problems:

  • Implement the recommended storage conditions: lyophilized (-20°C/-80°C) or in 50% glycerol

  • Avoid repeated freeze-thaw cycles

  • Consider protein stabilizing additives (glycerol, sucrose, BSA)

  • Follow reconstitution protocols to maintain the 0.1-1.0 mg/mL concentration range

• Functional activity assessment:

  • Develop robust activity assays based on known interactions

  • Compare activity with mammalian TIPE1

  • Include positive controls in functional assays

  • Consider co-factors that might be required for activity

How can researchers effectively detect and quantify endogenous tnfaip8l1 expression in zebrafish tissues?

For accurate detection and quantification of endogenous tnfaip8l1 in zebrafish tissues:

Methodological Approach Table:

When using antibodies, cross-reactivity testing is crucial as the TIPE family members share 75% amino acid sequence similarities . Researchers should consider developing zebrafish-specific antibodies if cross-reactivity with other TIPE family members is observed.

What are the most promising applications of tnfaip8l1 research in understanding human diseases?

The translational potential of zebrafish tnfaip8l1 research for human disease understanding includes:

• Cancer research applications:

  • Investigation of tumor suppressor mechanisms based on mammalian TIPE1's role in promoting apoptosis in liver cancer and hematological diseases

  • Understanding context-dependent functions, as TIPE1 has oncogenic properties in cervical cancer

  • Development of targeted therapeutics based on TIPE1 pathways

  • Zebrafish models for high-throughput drug screening targeting TIPE1-related pathways

• Inflammatory disease models:

  • Study of inflammatory regulation through TNF-α and NF-κB pathways

  • Assessment of therapeutic interventions in inflammatory conditions

  • Development of biomarkers for inflammatory disease progression

  • Insight into evolutionary conservation of inflammatory regulation

• Developmental biology applications:

  • Understanding cell survival mechanisms during development

  • Tissue-specific functions based on differential expression patterns

  • Interaction with conserved developmental pathways

  • Regenerative medicine applications based on apoptosis regulation

Researchers should consider both the conserved and divergent aspects of tnfaip8l1 function when translating findings from zebrafish to human applications, especially given the context-dependent functions observed in mammalian TIPE1.

What techniques are emerging for studying the structural biology of tnfaip8l1 and its interactions with binding partners?

Advanced structural biology approaches for investigating tnfaip8l1:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of protein complexes in near-native states

  • Can resolve conformational changes upon ligand binding

  • Particularly useful for membrane-associated complexes

  • Minimal sample preparation compared to crystallography

• Integrative structural biology:

  • Combines multiple techniques (X-ray crystallography, NMR, SAXS)

  • Creates comprehensive structural models

  • Addresses limitations of individual methods

  • Incorporates dynamic information

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein-protein interaction surfaces

  • Identifies conformational changes upon binding

  • Requires less sample than traditional structural methods

  • Works with challenging proteins resistant to crystallization

• Computational approaches:

  • Molecular dynamics simulations of tnfaip8l1 interactions

  • Homology modeling based on other TIPE family structures

  • Virtual screening for potential binding partners

  • Integration of experimental constraints with in silico predictions

Researchers should leverage the known sequence information (186 amino acids) and compare with the structural data available for other TIPE family members to generate preliminary models for experimental validation .