Recombinant Chicken E3 ubiquitin-protein ligase RNF185 (RNF185)

What is RNF185 and what are its key structural domains?

RNF185 is an evolutionarily conserved E3 ubiquitin ligase found in vertebrates. Human RNF185 is a 21 kDa protein characterized by a C3HC4 RING domain and two transmembrane (TM) domains, designated as TM1 and TM2. The RING domain is essential for its ubiquitin ligase activity, while the TM domains are critical for proper subcellular localization. Based on SMART prediction analysis, these domains appear to be conserved across species, suggesting similar structural organization in chicken RNF185 .

The functional significance of these domains has been demonstrated through mutational studies. When either the RING domain is mutated (RNF185-RM) or the TM domains are deleted (RNF185-TM), the protein loses its characteristic activities, indicating that both catalytic function and proper localization are essential for its biological roles .

Where is RNF185 primarily localized in cellular compartments?

RNF185 demonstrates dual localization patterns depending on cellular context:

• Mitochondrial localization: In human cells, RNF185 localizes predominantly to the mitochondrial outer membrane. This has been conclusively demonstrated through:

  • Colocalization with MitoTracker Red in confocal microscopy

  • Overlap with DsRed2-Mito in transfected cells

  • Enrichment in mitochondrial fractions during differential centrifugation

• Endoplasmic reticulum association: RNF185 also functions as an ER-associated E3 ligase involved in ER-associated degradation (ERAD). This association is critical for its role in protein quality control .

The transmembrane domains, particularly TM2, play a decisive role in determining this subcellular localization. Mutation studies revealed that RNF185-TM2 proteins showed more mislocalization compared to RNF185-TM1 mutants, indicating TM2's greater importance for proper targeting .

How does RNF185 contribute to mitochondrial autophagy regulation?

RNF185 plays a significant role in regulating selective mitochondrial autophagy (mitophagy) through several mechanisms:

• Autophagy induction: Overexpression of RNF185 stimulates LC3II accumulation, a well-established marker of autophagosome formation. This effect is dependent on both intact RING and TM domains .

• Substrate-specific ubiquitination: RNF185 polyubiquitinates BNIP1 (Bcl-2 Nineteen kilodalton Interacting Protein 1) through K63-based ubiquitin linkage. This interaction requires the TM domains of RNF185 and occurs at the mitochondria .

• Autophagy receptor recruitment: The polyubiquitinated BNIP1 recruits autophagy receptor p62, which simultaneously binds both ubiquitin and LC3, creating a bridge between ubiquitination and autophagy machinery .

• Mitochondrial mass regulation: Cells expressing high levels of RNF185 show dramatic loss of MitoTracker Red staining, indicating decreased mitochondrial mass, which correlates with enhanced mitochondrial degradation through autophagy .

This pathway represents a novel mechanism for modulating mitochondrial homeostasis through selective autophagy, potentially playing important roles in cellular stress responses and quality control.

What is the relationship between RNF185 and ER-associated degradation (ERAD)?

RNF185 functions as a key ERAD E3 ligase with specific targets and mechanisms:

This specificity for CFTR makes the RNF185/RNF5 module a potential therapeutic target for cystic fibrosis treatment, where increasing the stability of CFTRΔF508 could provide clinical benefit .

How does RNF185 interact with the VCP/p97 system in protein quality control?

RNF185 demonstrates significant interaction with the VCP/p97 machinery, a key component of cellular protein quality control:

• Co-factor interactions: RNF185 associates with the soluble VCP/p97 cofactors UFD1 and NPLOC4, supporting its role in ERAD-related functions. This interaction pattern is consistent with established ERAD E3 ligases like gp78/AMFR and Hrd1 .

• Enrichment analysis: Comparative analysis of VCP/p97 spectral counts placed RNF185 within the upper quartile of E3 ligases that enrich this AAA ATPase, alongside known ERAD E3s .

• Non-canonical recruitment: Interestingly, neither RNF185 nor its high-confidence interacting proteins (HCIPs) contain canonical VCP/p97 binding domains or motifs, suggesting either:

  • An unidentified binding factor mediates the interaction

  • A non-canonical or cryptic VCP/p97 binding motif exists in RNF185

  • VCP/p97 is recruited indirectly through ubiquitin-binding cofactors if RNF185 undergoes constitutive auto-ubiquitination

This interaction with the VCP/p97 system positions RNF185 as an important component in the extraction and processing of ubiquitinated substrates during cellular quality control operations.

What role does RNF185 play in calcium homeostasis and ER stress responses?

Recent research has revealed connections between RNF185 and calcium homeostasis through specific protein interactions:

• Calcium-regulatory protein associations: RNF185 enriches for TMUB1/TMUB2 and TMEM259/Membralin, proteins involved in cellular calcium signaling. These interactions were validated through co-expression and pulldown experiments with S-tagged HCIPs .

• ER stress involvement: These associated proteins are linked to homeostatic maintenance of ER Ca²⁺ levels related to ER stress responses and apoptotic regulation .

• Functional implications: The interaction with proteins like TMEM259/Membralin, which is linked to motor neuron survival, suggests that RNF185 may have neuroprotective functions through its role in calcium homeostasis regulation .

These findings highlight an emerging role for RNF185 in coordinating calcium signaling and ER stress responses, expanding its known functions beyond protein degradation pathways.

What expression systems are optimal for producing recombinant chicken RNF185?

Producing functional recombinant chicken RNF185 requires careful consideration of expression systems due to its membrane association and E3 ligase activity:

For functional studies of chicken RNF185, mammalian expression systems (particularly avian cell lines) would likely provide the most physiologically relevant protein, while E. coli systems may be sufficient for structural studies of soluble domains (e.g., the RING domain alone).

How can researchers assess the E3 ligase activity of recombinant RNF185 in vitro?

Multiple complementary approaches can be used to evaluate the E3 ligase activity of recombinant chicken RNF185:

• Self-ubiquitination assay: RNF185 demonstrates intensive polyubiquitination activity that can be measured as a proxy for its catalytic function. This activity is RING domain-dependent and requires proper subcellular localization .

  Protocol outline:

  • Incubate purified RNF185 with E1, E2 enzyme, ATP, and ubiquitin

  • Analyze reaction products by SDS-PAGE and Western blotting with anti-ubiquitin antibodies

  • Compare wild-type RNF185 with RING domain mutant (RNF185-RM) as a negative control

  • The TM-deleted mutant (RNF185-TM) should also show significantly reduced activity

• Substrate-specific ubiquitination: Based on known human RNF185 substrates like BNIP1, researchers can test ubiquitination of chicken orthologs of these proteins .

• Ubiquitin linkage analysis: Determining whether chicken RNF185 creates K63-linked chains (as seen with human RNF185) using ubiquitin mutants (K63R) or linkage-specific antibodies .

• E2 enzyme screening: Testing various E2 conjugating enzymes to identify optimal partners for chicken RNF185, which may differ from those preferred by human RNF185.

For all assays, it is critical to include appropriate controls, including reactions without ATP, without E1/E2 enzymes, or with catalytically inactive RNF185 mutants to confirm the specificity of the observed ubiquitination.

What approaches can be used to identify novel substrates of chicken RNF185?

Discovering novel substrates of chicken RNF185 requires integrating multiple complementary techniques:

• Protein interaction screening: RNF185 has been shown to interact with specific partners like BNIP1 and ATG5 in human cells. Similar co-immunoprecipitation studies with chicken RNF185 can identify potential binding partners .

• Domain-based interaction mapping: Studies with human RNF185 revealed that:

  • Binding to BNIP1 is dependent on their TM domains

  • The RING domain is not required for association with BNIP1

  • RNF185 binds to the coiled-coil (CC) domain of BNIP1
    This knowledge can guide targeted searches for chicken RNF185 substrates with similar structural features .

• Comparative proteomics: Analyzing changes in the ubiquitinome following RNF185 overexpression or knockdown in chicken cell lines. This can be enhanced by:

  • Using proteasome inhibitors to stabilize ubiquitinated proteins

  • Enriching for ubiquitinated peptides using anti-K-ε-GG antibodies

  • Quantitative mass spectrometry to identify enriched proteins

• Functional screening: Identifying proteins whose stability is regulated by RNF185 through pulse-chase experiments in the presence and absence of RNF185.

• VCP/p97 interaction analysis: Given RNF185's association with VCP/p97 and its cofactors, proteins that are co-regulated by both RNF185 and VCP/p97 represent promising substrate candidates .

Each approach has strengths and limitations, so integrating data from multiple methods provides the most robust substrate identification strategy.

How conserved is RNF185 across species, and what does this suggest about its function?

RNF185 demonstrates significant evolutionary conservation across vertebrates, providing insights into its fundamental biological importance:

• Domain conservation: The characteristic C3HC4 RING domain and two transmembrane domains (TM1 and TM2) are preserved across species, suggesting these structural features are essential for RNF185 function .

• Sequence homology: Alignment analysis shows that human RNF185 shares homology with RNF5, indicating they likely evolved from a common ancestor. This homology extends to functional overlap, as both target similar substrates like CFTR .

• Functional conservation: The dual roles of RNF185 in mitochondrial and ER quality control appear to be maintained across species, suggesting these are ancient and fundamental cellular processes requiring tight regulation .

• Species-specific variations: While core functions are conserved, species-specific adaptations likely exist, particularly in substrate recognition regions. These variations may reflect differences in cellular physiology or environmental adaptations.

The high degree of conservation suggests that findings from human RNF185 studies can provide valuable insights for research on chicken RNF185, while acknowledging potential species-specific differences in regulation and substrate specificity.

What are the key differences between chicken and human RNF185 that researchers should consider?

Researchers working with chicken RNF185 should be aware of several potential differences from the human ortholog:

• Expression patterns: While expression data for chicken RNF185 is limited, tissue-specific expression patterns may differ from those observed in humans. This could impact experimental design when studying tissue-specific functions.

• Substrate specificity: Even with conserved domains, subtle sequence variations may affect substrate recognition. Key human substrates like BNIP1 and CFTR should be validated in chicken systems rather than assumed.

• Interaction partners: The protein interaction network surrounding RNF185 may differ between species. Human RNF185 interacts with proteins like UFD1, NPLOC4, and TMEM259, but these interactions require verification in chicken cells .

• Regulatory mechanisms: Transcriptional and post-translational regulation of RNF185 may vary between species, affecting its expression levels and activity under different conditions.

• Antibody cross-reactivity: Commercial antibodies developed against human RNF185 may have variable cross-reactivity with chicken RNF185, necessitating validation or the development of chicken-specific reagents .

Careful comparative studies between human and chicken RNF185 will help identify both conserved functions and species-specific adaptations, enhancing the translational value of research findings.