Recombinant Danio rerio RING finger protein 170 (rnf170)

What is RNF170 and what is its role in cellular function?

RNF170 is a RING finger protein that functions as an E3 ubiquitin ligase, mediating the covalent attachment of ubiquitin to target proteins . The zebrafish ortholog shares significant sequence homology with human RNF170, suggesting conserved functionality. As part of the RING finger protein family, RNF170 contains a characteristic RING domain that forms a RING finger structure when combined with zinc atoms, providing a structural basis for interaction with E2 enzymes in the ubiquitination process . This post-translational modification system regulates thousands of proteins and is fundamentally important in eukaryotic cellular processes including protein degradation, signaling, trafficking, and quality control.

RNF170 is particularly noteworthy for its involvement in neurodegenerative pathways, with mutations in the gene linked to autosomal dominant sensory ataxia and hereditary spastic paraplegia . The protein appears to be evolutionarily conserved, indicating its functional importance across species.

What expression systems are used for producing recombinant Danio rerio RNF170?

E. coli is the predominant expression system used for recombinant Danio rerio RNF170 production, offering advantages in cost-effectiveness, scalability, and rapid growth . The full-length protein (266 amino acids) can be successfully expressed with an N-terminal His-tag to facilitate purification . This bacterial expression system is particularly suitable for structural and functional studies that require substantial quantities of purified protein.

The recombinant production typically involves:

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

• Transformation into a suitable E. coli strain

• Induction of protein expression

• Cell lysis and protein extraction

• Affinity chromatography using the His-tag for purification

Alternative eukaryotic expression systems may be considered for specialized applications requiring post-translational modifications, though these are not documented in the current literature for zebrafish RNF170.

What are the optimal storage and handling conditions for recombinant RNF170?

Proper storage and handling of recombinant RNF170 is critical for maintaining protein integrity and activity. The recommended protocol includes:

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

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

• Add glycerol to a final concentration of 5-50% (50% being standard) before aliquoting for long-term storage

• Store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they can compromise protein structure and function

Tris/PBS-based buffer with 6% trehalose at pH 8.0 is typically used as a storage buffer . Trehalose helps maintain protein stability during freeze-thaw cycles by preventing protein aggregation and denaturation.

How conserved is RNF170 between zebrafish and humans?

RNF170 exhibits significant conservation between zebrafish and humans, particularly in functionally important domains. This conservation provides the rationale for using zebrafish as a model organism to study RNF170-related human diseases .

Key observations regarding conservation include:

• The RING domain, critical for E3 ligase function, shows high sequence conservation

• Mutations identified in human disease-causing variants often affect residues that are conserved in zebrafish

• The functional conservation is demonstrated by the fact that mutations in RNF170 cause similar phenotypes in both humans and zebrafish models

This evolutionary conservation makes zebrafish an excellent model for studying the function of RNF170 and the pathogenic mechanisms of RNF170-related human diseases.

What methodologies are most effective for studying RNF170 function in zebrafish models?

Multiple complementary approaches have proven effective for investigating RNF170 function in zebrafish:

• Morpholino-mediated knockdown: Antisense morpholinos targeting exon-intron junctions (e.g., E3MO and E4MO) can effectively reduce RNF170 expression by causing inappropriate splicing and premature stop codons . Validation via RT-PCR using primers designed to detect altered splicing products is essential for confirming knockdown efficiency.

• mRNA microinjection: Wild-type or mutant RNF170 mRNA can be microinjected into zebrafish embryos to study the effects of overexpression or to perform rescue experiments in knockdown models . This approach has been instrumental in confirming the pathogenicity of human RNF170 mutations.

• CRISPR/Cas9 genome editing: Although not explicitly mentioned in the search results, CRISPR/Cas9 technology represents a more precise approach for generating stable RNF170 knockout or knock-in models in zebrafish.

• Behavioral and morphological phenotyping: Assessment of motor function, sensory responses, and spinal cord development can reveal functional consequences of RNF170 disruption that parallel human disease phenotypes.

• Biochemical assays: In vitro ubiquitination assays using recombinant RNF170 can identify substrates and characterize enzymatic activity under various conditions.

Each methodology offers unique advantages, and combining multiple approaches provides the most comprehensive understanding of RNF170 function.

How can morpholino knockdown be optimized for studying RNF170 in zebrafish embryos?

Optimizing morpholino (MO) knockdown of RNF170 in zebrafish requires careful consideration of several key parameters:

• Target site selection: Design morpholinos targeting exon-intron boundaries (splice-blocking MOs) as demonstrated with E3MO and E4MO, which target different splice sites in the rnf170 gene . This strategy causes intron retention or exon skipping, resulting in frameshift and premature stop codons.

• Validation of knockdown efficiency:

  • Design primers spanning exon-intron boundaries and perform RT-PCR to detect altered splicing products

  • Quantify the relative amplicon intensity between expected products in control versus morphant embryos

  • Normalize against housekeeping genes (e.g., GAPDH) to ensure accurate quantification

• Dose optimization: Titrate morpholino concentrations to identify the minimum dose that achieves significant knockdown while minimizing off-target effects.

• Controls:

  • Include control morpholino injections at equivalent concentrations

  • Perform rescue experiments with wild-type rnf170 mRNA to confirm specificity

  • Use multiple non-overlapping morpholinos (such as E3MO and E4MO) to validate phenotypes

• Timing considerations: Assess knockdown at appropriate developmental stages (e.g., 48 hpf) based on the temporal expression pattern of rnf170 .

This optimized approach enhances the reliability and specificity of RNF170 knockdown studies in zebrafish embryos.

What phenotypic consequences result from RNF170 mutations in zebrafish models?

Zebrafish with disrupted RNF170 function exhibit phenotypes that parallel human neurological disorders associated with RNF170 mutations:

• Developmental abnormalities: Microinjection of mutant RNF170 mRNA into zebrafish embryos causes dominant disruption of normal embryonic development .

• Neurological defects: Consistent with the role of RNF170 in human neurological disorders, zebrafish models show deficits in:

  • Motor function

  • Sensory processing

  • Neuronal development and maintenance

• Cellular pathology: Molecular and cellular alterations include:

  • Abnormal ubiquitination patterns

  • Protein aggregation

  • Defects in calcium homeostasis

  • Axonal degeneration reminiscent of hereditary spastic paraplegia or sensory ataxia

These phenotypes provide valuable insights into the pathogenic mechanisms underlying human neurological disorders associated with RNF170 mutations and establish zebrafish as a relevant model for studying these conditions.

How can recombinant RNF170 be used to study its E3 ubiquitin ligase activity in vitro?

Recombinant RNF170 provides a powerful tool for investigating its E3 ubiquitin ligase activity through several in vitro approaches:

• Ubiquitination assays: Reconstitute the ubiquitination cascade in vitro using:

  • Purified recombinant RNF170

  • E1 activating enzyme

  • Appropriate E2 conjugating enzymes

  • Ubiquitin (wild-type or tagged variants)

  • ATP

  • Potential substrate proteins

• Binding assays: Characterize interactions between:

  • RNF170 and E2 enzymes using pull-down assays

  • RNF170 and potential substrates using co-immunoprecipitation or surface plasmon resonance

  • Different domains of RNF170 and their binding partners

• Structure-function analysis:

  • Generate mutant versions of RNF170 with alterations in key domains

  • Analyze how mutations affect E3 ligase activity and substrate recognition

  • Perform comparative analysis of zebrafish RNF170 with human orthologs

• High-throughput screening:

  • Develop fluorescence-based ubiquitination assays using recombinant RNF170

  • Screen for small molecule modulators of RNF170 activity

  • Identify novel substrates using protein arrays

These in vitro approaches complement in vivo studies and provide mechanistic insights into RNF170 function at the molecular level.

What are the implications of RNF170 research for understanding human neurodegenerative disorders?

Research on RNF170 has significant implications for understanding and potentially treating human neurodegenerative disorders:

• Disease mechanisms: Studies in zebrafish have established that mutations in RNF170 are causal for:

  • Autosomal dominant sensory ataxia, characterized by degeneration of the posterior columns of the spinal cord

  • Hereditary spastic paraplegia, affecting upper motor neurons

• Molecular pathways: RNF170 research has revealed involvement in several key pathways disrupted in neurodegenerative conditions:

  • Protein quality control through the ubiquitin-proteasome system

  • Calcium homeostasis

  • Axonal maintenance and transport

  • Endoplasmic reticulum function

• Therapeutic potential: Identifying the precise mechanisms of RNF170-mediated neurodegeneration opens avenues for targeted therapeutic interventions:

  • Modulation of RNF170 enzymatic activity

  • Restoration of downstream pathways affected by RNF170 dysfunction

  • Gene therapy approaches to correct pathogenic mutations

• Biomarker development: RNF170 and its substrates may serve as biomarkers for:

  • Disease diagnosis

  • Monitoring disease progression

  • Evaluating therapeutic efficacy

The zebrafish model provides a valuable system for both understanding disease mechanisms and screening potential therapeutic compounds .

How do experimental approaches for studying RNF170 in zebrafish compare with mammalian models?

Zebrafish offer unique advantages and limitations relative to mammalian models for RNF170 research:

The most comprehensive approach combines the advantages of both systems:

• Initial high-throughput screening and mechanistic studies in zebrafish

• Validation and more complex analysis in mammalian models

• Translation of findings to human patient samples and clinical studies

What are the challenges in identifying RNF170 substrates and how can they be overcome?

Identifying the physiological substrates of RNF170 presents several challenges that require specialized approaches:

• Challenges:

  • Transient enzyme-substrate interactions

  • Rapid degradation of ubiquitinated substrates

  • Context-dependent ubiquitination

  • Redundancy in the ubiquitin system

  • Technical limitations in detecting ubiquitination events in vivo

• Methodological solutions:

  • Proteasome inhibition: Treat cells with proteasome inhibitors to stabilize ubiquitinated proteins

  • Tandem ubiquitin binding entities (TUBEs): Use these tools to enrich for ubiquitinated proteins

  • Proximity labeling: Employ BioID or APEX2 fused to RNF170 to identify proteins in close proximity

  • Comparative proteomics: Compare protein abundance in wild-type versus RNF170-deficient models

  • In vitro ubiquitination assays: Screen potential substrates using purified recombinant RNF170

  • Domain-specific interactions: Map binding domains to identify interaction motifs

  • Cross-linking mass spectrometry: Capture transient interactions between RNF170 and substrates

• Zebrafish-specific approaches:

  • Temporal analysis of the zebrafish proteome during development in RNF170 morphants

  • Tissue-specific proteomics in neuronal populations affected by RNF170 mutations

  • Parallel analysis in zebrafish and human cell models to identify conserved substrates

These complementary approaches overcome individual limitations and increase confidence in identified substrates.

How can zebrafish RNF170 models contribute to drug discovery for related human diseases?

Zebrafish models of RNF170 dysfunction offer unique advantages for drug discovery related to human neurodegenerative diseases:

• High-throughput screening platforms:

  • Embryo-based phenotypic screens can assess thousands of compounds

  • Automation of embryo handling, compound delivery, and phenotype assessment

  • Ability to identify compounds that suppress RNF170-related developmental or neurological phenotypes

• Target validation:

  • Confirmation of RNF170 as a therapeutic target through genetic manipulation

  • Validation of downstream pathways as alternative targeting strategies

  • Comparison of chemical and genetic suppression of phenotypes

• Mechanistic insights:

  • Identification of compounds that modulate RNF170 enzymatic activity

  • Discovery of molecules that affect RNF170-substrate interactions

  • Compounds that compensate for loss of RNF170 function

• Translational pathway:

  • Initial screening in zebrafish models

  • Validation in mammalian cell models

  • Testing in rodent models of RNF170-related diseases

  • Clinical development for hereditary spastic paraplegia and sensory ataxia

• Advantages over alternative approaches:

  • Whole-organism context that includes blood-brain barrier considerations

  • Assessment of both efficacy and toxicity simultaneously

  • Cost-effective compared to mammalian models

  • Rapid development timeline from target identification to lead compound

This integrated drug discovery approach leverages the unique advantages of zebrafish models while maintaining relevance to human disease.