Recombinant Danio rerio E3 ubiquitin-protein ligase RNF185 (rnf185)

What is RNF185 and what are its primary functions?

RNF185 is a RING domain-containing E3 ubiquitin ligase that coordinates substrate recognition, export, and proteasome targeting as a key component of endoplasmic reticulum-associated degradation (ERAD) . Its primary functions include:

• Targeting specific proteins for ubiquitination and subsequent proteasomal degradation

• Regulating ER protein quality control mechanisms

• Participating in the co-translational degradation of proteins like CFTR

• Contributing to antiviral innate immune responses

In humans, RNF185 has been demonstrated to preferentially associate with the E2 ubiquitin-conjugating enzymes UBE2J1 and UBE2J2, suggesting conserved enzymatic partnerships across species .

How is RNF185 structurally organized?

RNF185 contains several key structural features:

• A RING finger domain essential for E3 ubiquitin ligase activity

• Transmembrane domains that anchor it to the endoplasmic reticulum membrane

• Cytoplasmic regions that interact with E2 ubiquitin-conjugating enzymes

• Regions that facilitate specific substrate recognition

The RING domain is particularly critical as mutations in this region can abolish the protein's ubiquitin ligase activity, as demonstrated in studies with human RNF185 .

What are the known cellular localizations of RNF185?

RNF185 is predominantly localized to the endoplasmic reticulum (ER) membrane . Subcellular localization studies have demonstrated that:

• RNF185 contains transmembrane domains that anchor it to the ER membrane

• It co-localizes with other ER markers and ERAD components

• It forms functional complexes with other ER-resident proteins

• It may partially localize to ER-Golgi intermediate compartments during certain cellular processes

Fluorescent tagging experiments with human RNF185 have confirmed its ER localization, where it participates in protein quality control and degradation pathways .

What are the common model systems used to study RNF185?

Several model systems have been employed to study RNF185 function:

When working specifically with Danio rerio RNF185, researchers typically utilize embryos for developmental studies and isolated tissues for functional analyses of the native protein within its physiological context.

How does RNF185 interact with other components of the ERAD machinery?

RNF185 forms complex networks with other ERAD components to facilitate efficient protein degradation:

• RNF185 and RNF5 can function synergistically in the degradation of substrates like CFTR, with both co-translational and post-translational mechanisms

• Interaction mapping has identified associations between RNF185 and UBL domain-containing proteins TMUB1/TMUB2, which may serve as adaptors in the ERAD pathway

• RNF185 co-precipitates with other E3 ligases including RNF170 and RNF5, suggesting coordinated or sequential ubiquitination as part of ERAD

• The ERLIN1/2 heterodimer interacts with RNF185 and may serve as a bridge for larger hetero-oligomeric E3 complexes

These interactions highlight RNF185's integration within a larger quality control network at the ER membrane, working cooperatively with other E3 ligases to target specific substrates.

What is the role of RNF185 in viral infection responses?

Recent research has uncovered important functions of RNF185 in antiviral responses:

• RNF185 regulates the stability of the SARS-CoV-2 envelope protein through ubiquitination and degradation

• RNF185 co-localizes with the SARS-CoV-2 envelope protein at the endoplasmic reticulum

• Depletion of RNF185 significantly increases SARS-CoV-2 viral titer in cellular models, suggesting its role in restricting viral replication

• RNF185 has been implicated in the cGAS-mediated innate immune response upon HSV-1 infection, catalyzing 'Lys-27'-linked polyubiquitination of cGAS

This dual role in viral protein degradation and innate immune signaling positions RNF185 as a potentially important regulator of host-pathogen interactions, making it a target of interest for antiviral therapeutic development.

How is RNF185 expression regulated during cellular stress?

RNF185 expression responds to various cellular stressors, particularly those affecting the endoplasmic reticulum:

• Studies have examined RNF185 expression during the unfolded protein response (UPR) using tunicamycin treatment, which induces ER stress

• RNF185 itself appears to protect cells from ER stress-induced apoptosis

• The regulation of RNF185 may involve autoubiquitination and proteasome-dependent degradation, similar to other RING E3 ligases

• Expression patterns during development and in different tissues remain to be fully characterized, particularly in zebrafish models

Understanding the regulation of RNF185 could provide insights into how cells modulate protein quality control mechanisms during stress conditions.

What are the known substrates of RNF185 and their degradation mechanisms?

Several substrates of RNF185 have been identified through various experimental approaches:

RNF185 shows substrate specificity, as it does not control the degradation of all classical ERAD model substrates . This selectivity suggests specialized roles in quality control and regulatory pathways that may be conserved in the Danio rerio ortholog.

What are the recommended approaches for expressing and purifying recombinant Danio rerio RNF185?

Based on protocols used for related proteins, the following approaches are recommended:

• Expression Systems:

  • E. coli BL21(DE3) for high yield of the cytosolic domain

  • Insect cells (Sf9 or Hi5) for full-length protein with post-translational modifications

  • Mammalian cells (HEK293) for properly folded protein with native modifications

• Purification Strategy:

  • Affinity chromatography using His-tag or GST-tag

  • Size exclusion chromatography to ensure homogeneity

  • Ion exchange chromatography for higher purity

• Buffer Considerations:

  • Include reducing agents (DTT or β-mercaptoethanol) to maintain RING domain integrity

  • Consider detergents for full-length protein with transmembrane domains

  • Add protease inhibitors to prevent degradation during purification

Adapting protocols used for human RNF185 while accounting for species-specific differences is recommended for optimal results with the zebrafish protein.

How can in vitro ubiquitination assays be optimized for RNF185?

In vitro ubiquitination assays require careful optimization:

• Components Required:

  • Purified recombinant RNF185 (E3)

  • E1 activating enzyme (typically UBA1)

  • E2 conjugating enzymes (preferably UBE2J1 and UBE2J2 based on known preferences)

  • Ubiquitin (consider using tagged versions for detection)

  • ATP regeneration system

  • Target substrate (if known)

• Optimization Parameters:

  • Temperature (typically 30-37°C)

  • Reaction time (15 min to 2 hours)

  • Component concentrations

  • Buffer composition (pH, salt concentration)

• Controls:

  • RING domain mutant RNF185 (negative control)

  • Omission of ATP (negative control)

  • Known E3 ligase with established activity (positive control)

Detection methods include western blotting with anti-ubiquitin antibodies, using tagged ubiquitin (His, FLAG, or biotin), or mass spectrometry for identifying ubiquitination sites.

What are the best approaches to identify novel RNF185 substrates and interacting partners?

Several complementary approaches can be employed:

• Proteomics-Based Methods:

  • Immunoprecipitation followed by mass spectrometry (IP-MS)

  • Proximity labeling techniques (BioID or APEX)

  • Ubiquitin remnant profiling in RNF185 overexpression or knockout systems

  • Comparative proteomic workflow as used for human ER-E3 ligases

• Genetic Screens:

  • CRISPR-Cas9 screens to identify genetic interactions

  • Yeast two-hybrid screening with the cytosolic domains

  • Suppressor/enhancer screens in zebrafish models

• Biochemical Validation:

  • Co-immunoprecipitation to confirm direct interactions

  • In vitro and in vivo ubiquitination assays

  • Protein stability assays in the presence/absence of RNF185

This multi-faceted approach can help build a comprehensive interactome of Danio rerio RNF185, potentially revealing conserved and species-specific functions.

How can zebrafish models be effectively utilized to study RNF185 function in vivo?

Zebrafish provide an excellent vertebrate model system for studying RNF185:

• Genetic Manipulation Approaches:

  • Morpholino knockdown for transient loss-of-function studies

  • CRISPR-Cas9 for generating stable knockout or knock-in lines

  • Transgenic overexpression using tissue-specific promoters

  • Heat shock-inducible expression systems for temporal control

• Phenotypic Analysis:

  • Development and morphology assessment

  • Tissue-specific defects in ER structure or function

  • Response to stressors (e.g., tunicamycin for ER stress)

  • Susceptibility to viral infection

• Molecular Analysis:

  • In situ hybridization to determine expression patterns

  • Quantitative PCR to measure transcript levels

  • Western blotting to assess protein levels

  • Immunohistochemistry for localization studies

Zebrafish models can provide valuable insights into the developmental and physiological roles of RNF185 that may be difficult to observe in cell culture systems.

How can issues with RNF185 expression and solubility be addressed?

Common challenges and solutions include:

• Low Expression Levels:

  • Optimize codon usage for the expression system

  • Test different promoters or induction conditions

  • Consider using fusion partners (MBP, SUMO) to enhance expression

  • Examine expression at lower temperatures (16-18°C)

• Poor Solubility:

  • Express soluble domains separately from transmembrane regions

  • Use mild detergents for membrane protein extraction (DDM, CHAPS)

  • Include stabilizing agents (glycerol, arginine) in buffers

  • Consider using insect or mammalian expression systems

• Protein Degradation:

  • Include protease inhibitors during all purification steps

  • Perform purification at 4°C

  • Minimize freeze-thaw cycles

  • Add reducing agents to prevent RING domain oxidation

Systematic optimization of these parameters can significantly improve yield and quality of recombinant Danio rerio RNF185.

What are common pitfalls in interpreting RNF185 functional assays?

Researchers should be aware of several potential issues:

• Substrate Specificity Considerations:

  • Verify that observed ubiquitination is directly mediated by RNF185 using catalytically inactive mutants

  • Rule out indirect effects through other E3 ligases that may interact with RNF185

  • Consider that substrate recognition might differ between human and zebrafish orthologs

• Localization Artifacts:

  • Overexpression may lead to mislocalization

  • Fusion tags may interfere with proper targeting

  • Fixation methods can affect membrane protein localization

• Functional Redundancy:

  • Consider potential compensation by related E3 ligases (e.g., RNF5)

  • Validate findings using multiple approaches (knockdown, knockout, dominant negative)

  • Examine combined depletion of functionally related E3s

• Species-Specific Differences:

  • Functions established for human RNF185 may not be fully conserved in zebrafish

  • Consider environmental factors relevant to aquatic organisms

  • Validate findings across multiple model systems when possible

Careful experimental design and appropriate controls are essential for meaningful interpretation of RNF185 functional studies.

What are the emerging therapeutic applications targeting RNF185?

Several potential therapeutic applications are being explored:

• Cystic Fibrosis Treatment:

  • Small molecules that inhibit RNF185 could potentially stabilize CFTRΔF508, complementing existing therapeutics

  • Combined targeting of the RNF5/RNF185 module might be more effective than targeting either alone

• Antiviral Strategies:

  • Compounds that enhance RNF185-mediated degradation of viral proteins (like SARS-CoV-2 envelope) could have antiviral effects

  • Small-molecule binders that increase retention time of viral proteins in the ER could enhance degradation by RNF185

• Immunomodulation:

  • Given RNF185's role in regulating cGAS-STING pathway , targeting this interaction could modulate innate immune responses

• ER Stress-Related Conditions:

  • Modulation of RNF185 activity might protect against ER stress-induced apoptosis in relevant disease models

The development of specific modulators of RNF185 activity or its substrate interactions represents an exciting frontier in translational research.

What are key unresolved questions about RNF185 biology?

Several fundamental questions remain to be addressed:

• Developmental Roles:

  • How does RNF185 function during embryonic development?

  • Are there tissue-specific functions of RNF185 in Danio rerio?

  • What phenotypes result from complete loss of RNF185 function?

• Substrate Repertoire:

  • What is the complete set of physiological substrates?

  • How is substrate specificity determined at the molecular level?

  • Are there species-specific substrates in zebrafish versus mammals?

• Regulation Mechanisms:

  • How is RNF185 activity regulated post-translationally?

  • What signaling pathways modulate RNF185 function?

  • How does RNF185 coordinate with other E3 ligases in larger complexes?

• Evolutionary Conservation:

  • How conserved are RNF185 functions across vertebrates?

  • What domains or activities represent core functions versus species-specific adaptations?

Addressing these questions will provide deeper insights into the biological significance of RNF185 and potentially uncover novel therapeutic targets.