Recombinant Pongo abelii E3 ubiquitin-protein ligase RNF185 (RNF185)

What is the structural composition of Pongo abelii RNF185?

RNF185 is a RING domain-containing E3 ubiquitin ligase with 192 amino acids in its expression region. The full amino acid sequence is: MASKGPSASSSPENSSAGGPSGSSNGAGESGGQDSTFECNICLDTAKDAVISLCGHLFCWPCLHQWLETRPNRQVCPVCKAGISRDKVIPLYGRGSTGQQDPREKTPPRPQGQRPEPENRGGFQGFGFGDGGFQMSFGIGAFPFGIFATAFNINDGRPPPAVPGTPQYVDEQFLSRLFLF VALVIMFWLLIA. The protein contains characteristic cysteine and histidine residues in its RING finger motif that coordinate zinc ions, which is essential for its catalytic activity. The protein is identified in the UniProt database under accession number Q5RFK9 .

How does RNF185 function in the endoplasmic reticulum-associated degradation (ERAD) pathway?

RNF185 functions as an E3 ubiquitin ligase that selectively targets misfolded proteins for degradation via the ubiquitin-proteasome system. It specifically recognizes and ubiquitinates substrates like cystic fibrosis transmembrane conductance regulator (CFTR) and its mutant form CFTRΔF508. RNF185 primarily acts during co-translational degradation (as proteins are being synthesized) but also works in post-translational degradation when paired with its homolog RNF5. This RING-dependent activity is critical for maintaining protein quality control in the endoplasmic reticulum .

What are the optimal conditions for storing and handling recombinant RNF185?

Recombinant RNF185 should be stored in Tris-based buffer with 50% glycerol at -20°C for regular use or at -80°C for extended storage. Repeated freezing and thawing cycles should be avoided as they may compromise protein activity. For short-term work, aliquots can be maintained at 4°C for up to one week. The recombinant protein is typically available in 50 μg quantities, though custom amounts can be requested for specific experimental needs .

How can researchers clone and express RNF185 for functional studies?

RNF185 can be cloned from cDNA using high-fidelity reverse transcription. The following methodology has been validated:

• Extract total RNA from target cells (e.g., HEK293)

• Synthesize cDNA using a high-fidelity kit such as Transcriptor High Fidelity cDNA synthesis kit

• PCR-amplify using specific primers:

  • Forward: GAAGATCTGCAAGCAAGGGGCCCTCGGCC

  • Reverse: CCGCTCGAGTTAGGCAATCAGGAGCCAGAACATG

• Clone the amplified fragment into expression vectors (e.g., pcDNA3.1 FLAG) using BamHI/XhoI restriction sites

• Generate deletion mutants for domain studies using specific primers:

  • RNF185 ΔC (amino acids 1-176): Use reverse primer CCGCTCGAGTTAGCGTGACAGGAACTGCTCGTC

  • RNF185 ΔR (amino acids 94-192): Use forward primer GAAGATCTAGGGGCAGCACTGGGCAAC

This approach enables the expression of wild-type and mutant forms for comparative functional analyses .

What methods are effective for quantifying RNF185 expression in different tissues?

Quantitative PCR (qPCR) is the recommended method using the following species-specific primers:

Table 1: Validated primers for RNF185 expression analysis

For cellular experiments, GAPDH should be used as the reference gene, while 18S RNA and PPIA1 are recommended for tissue-specific expression analyses. This methodology ensures accurate normalization and comparison of RNF185 expression levels across different experimental conditions .

How does the RNF185-RNF5 functional module regulate CFTR degradation?

RNF185 and RNF5 form a cooperative E3 ligase module that controls CFTR degradation through complementary mechanisms. Current research indicates:

• RNF185, like RNF5, targets CFTR and CFTRΔF508 for co-translational degradation

• RNF5 primes CFTRΔF508 through initial ubiquitination during translation

• GP78 (another E3 ligase) subsequently elongates the ubiquitin chain to promote efficient degradation

• The combined depletion of both RNF185 and RNF5 dramatically blocks CFTRΔF508 degradation both during and after translation

• This functional redundancy and synergy suggests evolutionary importance in maintaining protein quality control

This coordinated activity positions the RNF5/RNF185 module as a potential therapeutic target for cystic fibrosis treatment, as modulating their activity could potentially increase the stability and function of CFTRΔF508 .

How is RNF185 expression regulated during cellular stress responses?

RNF185 expression undergoes significant modulation during the unfolded protein response (UPR), which is activated during endoplasmic reticulum stress. Experimental data demonstrates that treatment with tunicamycin (2 μg/ml), a known inducer of ER stress, alters RNF185 expression patterns. This regulation can be monitored by quantitative PCR alongside established UPR markers such as GRP78 (BiP). These changes in expression levels reflect the protein's role in helping cells adapt to protein folding stress conditions by facilitating the removal of misfolded proteins through the ERAD pathway. This regulatory mechanism represents an important adaptive response to maintain cellular homeostasis during ER stress .

What is the role of RNF185 in viral infection, particularly SARS-CoV-2?

RNF185 has been identified as a critical regulator of SARS-CoV-2 envelope protein stability through the following mechanisms:

• RNF185 and the SARS-CoV-2 envelope protein co-localize at the endoplasmic reticulum

• RNF185 promotes the degradation of the viral envelope protein through the ubiquitin-proteasome pathway

• CRISPR-Cas9 knockout of RNF185 increases SARS-CoV-2 envelope-eGFP expression by 2-3 fold in multiple cell lines

• Depletion of RNF185 significantly increases SARS-CoV-2 viral titers in cellular models

Table 2: Effect of RNF185 depletion on SARS-CoV-2 envelope protein levels

These findings suggest that RNF185 may play a protective role against SARS-CoV-2 infection by limiting viral protein availability. Modulation of this interaction represents a potential opportunity for novel antiviral therapeutic strategies .

How might targeting RNF185 benefit cystic fibrosis treatment strategies?

The RNF185/RNF5 E3 ligase module represents a promising therapeutic target for cystic fibrosis treatment based on the following research findings:

• Both RNF185 and RNF5 target CFTRΔF508 (the most common mutation in cystic fibrosis) for degradation

• Their combined activity significantly limits the amount of CFTRΔF508 that could potentially reach the cell surface

• Simultaneous depletion of both RNF185 and RNF5 dramatically stabilizes CFTRΔF508

• Inhibiting this E3 ligase module could potentially increase the stability and function of CFTRΔF508

Therapeutic approaches could include small molecule inhibitors targeting the RING domains, disrupting specific protein-protein interactions, or employing targeted proteolysis strategies. By allowing more CFTRΔF508 to avoid degradation, these approaches might complement existing cystic fibrosis therapeutics that aim to enhance the function of the limited amount of CFTRΔF508 that escapes ERAD .

How can researchers distinguish between the effects of RNF185 and other E3 ligases with similar functions?

Researchers should implement a multi-faceted experimental approach:

• Generate single and combination knockout cell lines (particularly RNF185 and RNF5) using CRISPR-Cas9 technology with at least 4 different sgRNAs per target

• Perform rescue experiments with wild-type and catalytically inactive mutant variants

• Compare degradation kinetics using pulse-chase experiments to distinguish co-translational versus post-translational effects

• Analyze ubiquitin chain topology on substrates using mass spectrometry (different E3 ligases may generate distinct ubiquitin linkage types)

• Conduct in vitro ubiquitination assays with purified components to confirm direct enzymatic activity

• Examine substrate specificity across multiple potential targets (CFTR, CFTRΔF508, and viral proteins like SARS-CoV-2 envelope)

This systematic approach will help delineate the specific contributions of RNF185 compared to other ERAD E3 ligases like RNF5 and GP78 .

What are the key considerations when studying RNF185 across different species?

When conducting comparative studies of RNF185 across species, researchers should consider:

• Sequence homology analysis focusing particularly on the RING domain and substrate-binding regions

• Expression pattern differences in various tissues and developmental stages

• Substrate specificity variations that may reflect species-specific adaptations

• Differential responses to cellular stressors like unfolded protein response induction

• Co-evolutionary relationships with key binding partners and substrates

• Potential differences in post-translational modifications that may regulate activity

RNF185 from Pongo abelii (Sumatran orangutan) serves as an important comparative model for understanding human RNF185 function, as primates often share significant functional conservation in protein quality control mechanisms .

How can RNF185 be studied in the context of SARS-CoV-2 variant evolution?

The study of RNF185 in the context of SARS-CoV-2 variants requires a specialized approach:

• Express envelope proteins from multiple SARS-CoV-2 variants (original, Alpha, Delta, Omicron) in cell culture systems

• Quantify the degradation kinetics of each variant's envelope protein in the presence and absence of RNF185

• Perform co-immunoprecipitation experiments to assess potential differences in binding affinity

• Identify key amino acid residues in the viral envelope that may have evolved to evade RNF185-mediated degradation

• Determine whether RNF185 depletion differentially affects viral titers across variants

• Explore the potential of RNF185 enhancers as pan-coronavirus therapeutic candidates

This research direction would provide insights into how the host-virus protein quality control interface contributes to viral evolution and pathogenicity, potentially leading to novel antiviral strategies .

What novel techniques could advance our understanding of RNF185's substrate selectivity?

Several cutting-edge methodologies could reveal new insights into RNF185 substrate selection:

• Proximity labeling approaches (BioID or TurboID fused to RNF185) to identify the complete interactome in living cells

• Global protein stability profiling after RNF185 manipulation to discover novel substrates

• Cryo-electron microscopy to determine the structural basis of substrate recognition

• Deep mutational scanning of both RNF185 and known substrates to map interaction interfaces

• Single-cell analysis of RNF185 activity using fluorescent reporters to understand cell-to-cell variability

• Organoid systems derived from patients with relevant diseases (e.g., cystic fibrosis) to study RNF185 in a physiologically relevant context

These approaches would overcome limitations of traditional biochemical methods and provide a systems-level understanding of how RNF185 contributes to protein quality control in different cellular contexts .