Recombinant Human E3 ubiquitin-protein ligase RNF185 (RNF185)

What is the basic structure of RNF185?

RNF185 is a 21 kDa protein containing a C3HC4 RING finger domain and two transmembrane domains (TM1 and TM2). The RING domain is essential for its ubiquitin E3 ligase activity, while the transmembrane domains are critical for its proper subcellular localization. RNF185 is evolutionarily conserved among vertebrates and shares approximately 70% sequence identity with RNF5, another E3 ubiquitin ligase . The structural integrity of both the RING domain and transmembrane domains is essential for RNF185's function, as mutations in either significantly impact its ubiquitin ligase activity and cellular localization .

Where is RNF185 primarily localized in cells?

RNF185 primarily localizes to both the endoplasmic reticulum (ER) and mitochondria. Subcellular localization studies using GFP-tagged RNF185 and confocal microscopy have shown clear overlap with MitoTracker Red, indicating mitochondrial localization. This has been further confirmed by differential centrifugation experiments showing endogenous RNF185 is most abundant in mitochondria-enriched fractions . Additionally, RNF185 has been identified as an ER membrane-associated protein involved in ERAD pathways . The transmembrane domains, particularly TM2, play crucial roles in determining this subcellular localization, as their mutation or deletion leads to mislocalization of the protein .

What are the major cellular pathways involving RNF185?

RNF185 participates in several critical cellular pathways:

• ER-associated degradation (ERAD): RNF185 targets misfolded proteins like CFTR and CFTRΔF508 for ubiquitination and subsequent proteasomal degradation .

• Mitochondrial autophagy: RNF185 regulates selective mitochondrial autophagy by mediating 'Lys-63'-linked polyubiquitination of BNIP1 .

• Innate immune signaling: RNF185 facilitates cGAS-mediated innate immune responses by catalyzing K27-linked polyubiquitination of cGAS .

• Viral protein regulation: RNF185 has been identified as a regulator of SARS-CoV-2 envelope protein stability .

• Cancer progression: RNF185 limits prostate cancer migration and metastasis through control of COL3A1 expression .

Each of these pathways represents a distinct functional role of RNF185, highlighting its versatility as a regulatory protein in cellular homeostasis and stress responses.

What are the recommended methods for studying RNF185 localization?

To study RNF185 localization, a combination of fluorescence microscopy and biochemical fractionation techniques is recommended:

• Fluorescence microscopy:

  • Express GFP-tagged RNF185 constructs in cell lines of interest

  • Co-stain with organelle-specific markers (e.g., MitoTracker Red for mitochondria, ER-Tracker for endoplasmic reticulum)

  • Use confocal microscopy to assess colocalization

  • For endogenous RNF185, use affinity-purified anti-RNF185 polyclonal antibodies with Alexa Fluor 488 secondary antibodies

• Subcellular fractionation:

  • Perform differential centrifugation to separate cellular compartments

  • Analyze fractions by western blotting using RNF185-specific antibodies

  • Include organelle-specific markers as controls (e.g., mitochondrial, ER, cytosolic markers)

• Domain mutation analysis:

  • Generate constructs with mutations in specific domains (RING domain, TM1, TM2)

  • Assess the impact on localization using the methods above

  • This approach helps determine which domains are essential for proper targeting

For optimal results, combine these approaches to confirm localization patterns and validate findings across multiple cell types.

How can researchers effectively detect RNF185-mediated ubiquitination?

To detect and characterize RNF185-mediated ubiquitination, researchers should employ the following methods:

• In vivo ubiquitination assays:

  • Co-transfect cells with plasmids expressing RNF185 (wild-type or mutants), target protein, and tagged ubiquitin

  • Perform immunoprecipitation using antibodies against the target protein

  • Detect ubiquitination by western blotting with anti-ubiquitin or anti-tag antibodies

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

• Chain-specific ubiquitination analysis:

  • Use ubiquitin mutants (K27, K48, K63 only) to determine the type of ubiquitin chains being formed

  • RNF185 has been shown to catalyze K27-linked chains on cGAS and K63-linked chains on BNIP1

• Protein stability assays:

  • Perform cycloheximide chase experiments to assess target protein half-life in the presence or absence of RNF185

  • Compare wild-type RNF185 with catalytically inactive mutants (e.g., RING domain mutants)

• Mass spectrometry:

  • For unbiased identification of ubiquitination sites on target proteins

  • Immunoprecipitate the target protein after co-expression with RNF185

  • Perform tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Look for the characteristic GG dipeptide signature on lysine residues

These complementary approaches provide robust evidence for RNF185-mediated ubiquitination and help characterize the functional consequences of this modification.

What genetic tools are most effective for studying RNF185 function?

Several genetic tools have proven effective for studying RNF185 function:

• RNA interference (RNAi):

  • siRNA or shRNA targeting RNF185

  • For rescue experiments, use RNAi-resistant RNF185 constructs with silent mutations in the targeted sequence

  • Example primer for human RNF185: 5'-CTGTCACGCCTCTTCCTATTTGT-3' (forward) and 5'-GCCCAGCATTAGGCAATCAG-3' (reverse)

• CRISPR-Cas9 gene editing:

  • For complete knockout of RNF185

  • Design multiple sgRNAs targeting RNF185 exons

  • Validate knockout by western blotting and functional assays

• Domain-specific mutants:

  • RING domain mutants (e.g., C39A) to disrupt E3 ligase activity

  • Transmembrane domain mutants or deletions to alter localization

  • These constructs help dissect the importance of specific domains for RNF185 function

• Fusion protein constructs:

  • GFP or RFP fusions for localization studies

  • FLAG, HA, or other epitope tags for immunoprecipitation experiments

  • Example: GFP-RNF185-WT, RNF185-RM (RING mutant), RNF185-TM (transmembrane domains deleted)

• Inducible expression systems:

  • Tetracycline-inducible systems for controlled expression of RNF185

  • Useful for studying dose-dependent and temporal effects

Each approach has specific advantages and limitations, and combining multiple techniques provides more robust insights into RNF185 function.

How does RNF185 contribute to SARS-CoV-2 infection?

RNF185 has been identified as a regulator of SARS-CoV-2 envelope protein stability, with significant implications for viral replication:

• Regulation of envelope protein degradation:

  • RNF185 targets the SARS-CoV-2 envelope protein for ubiquitin-dependent degradation

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

  • Depletion of RNF185 significantly increases SARS-CoV-2 envelope protein stability

• Impact on viral replication:

  • CRISPR-Cas9 knockout of RNF185 results in a 2- to 3-fold increase in SARS-CoV-2 viral titers in cellular models

  • This suggests RNF185 functions as an intrinsic antiviral factor by limiting envelope protein availability

• Variant specificity:

  • RNF185-mediated degradation affects envelope proteins from various SARS-CoV-2 clinical variants

  • The effect extends to SARS-CoV envelope protein but not MERS coronavirus envelope protein

• Mechanistic dependency:

  • The degradation process involves the ERAD complex, specifically requiring TMEM259

  • Depletion of TMEM259, a member of the ERAD complex required for RNF185 function, phenocopies RNF185 knockout

These findings suggest that modulating RNF185 activity or its interaction with the viral envelope protein could represent a novel strategy for antiviral therapeutics. Enhancing RNF185-mediated degradation of the envelope protein might reduce viral loads and disease severity.

What is known about RNF185's role in cancer progression?

RNF185 has emerged as a significant regulator of cancer progression, particularly in prostate cancer:

• Expression correlation with cancer progression:

  • Prostate tumor patient data analysis shows a negative correlation between RNF185 expression and cancer progression/metastasis

  • Lower RNF185 expression is associated with more aggressive disease

• Functional impact on cancer cell behavior:

  • RNF185 depletion in prostate cancer cell lines leads to:

    • Enhanced migration and invasion capabilities in vitro

    • Larger tumors and more frequent lung metastases in mouse models

• Molecular mechanism:

  • RNF185 controls the expression of COL3A1 (collagen type III alpha 1 chain)

  • Low RNF185 expression leads to upregulation of genes involved in epithelial-to-mesenchymal transition (EMT)

  • RNA-sequencing and Ingenuity Pathway Analysis identified wound-healing and cellular movement pathways as significantly upregulated in RNF185-depleted cells

• Therapeutic implications:

  • Co-inhibition of COL3A1 attenuates the enhanced migration and metastasis observed in RNF185 knockdown cells

  • This suggests targeting the RNF185-COL3A1 axis as a potential strategy to limit metastatic progression

These findings position RNF185 as a potential biomarker for prostate cancer progression and a gatekeeper of metastasis. The RNF185-COL3A1 regulatory axis represents a promising therapeutic target for interventions aimed at preventing or limiting metastatic spread.

How does RNF185 function in innate immune responses?

RNF185 plays a critical role in innate immune responses, particularly in the cGAS-STING pathway:

• Regulation of cGAS activity:

  • RNF185 catalyzes K27-linked polyubiquitination of cGAS at lysine residues K173 and K384

  • This modification enhances cGAS enzymatic activity rather than targeting it for degradation

  • RNF185-mediated ubiquitination promotes the production of 2'3'-cGAMP, the second messenger that activates STING

• Impact on antiviral responses:

  • Ectopic expression of RNF185 enhances IRF3-responsive gene expression after DNA stimulation

  • Knockdown of RNF185 impairs the induction of type I interferons and other antiviral genes

  • RNF185 depletion in bone marrow-derived macrophages (BMDMs) increases HSV-1 viral titers

• Virus-specific regulation:

  • RNF185 specifically regulates responses to cytosolic DNA and DNA viruses (like HSV-1)

  • It does not affect responses to RNA viruses (like Sendai virus)

  • This specificity is due to its action on cGAS, which is upstream of STING in the DNA sensing pathway

• Potential link to autoimmunity:

  • Systemic Lupus Erythematosus (SLE) patients display elevated expression of RNF185 mRNA

  • This suggests a potential role for RNF185 in autoimmune diseases characterized by aberrant responses to self-DNA

• Functional requirements:

  • The E3 ligase activity of RNF185 is essential for its function in innate immunity

  • RNF185 with a mutated RING domain (C39A) fails to enhance antiviral responses

These findings highlight RNF185 as a critical positive regulator of the cGAS-STING pathway and innate immune responses to DNA viruses, with potential implications for both infectious diseases and autoimmunity.

What proteins interact with RNF185 and how should these interactions be studied?

RNF185 engages in numerous protein-protein interactions central to its diverse functions. These interactions and recommended study methods include:

Key Interaction Partners:

Recommended Study Methods:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of RNF185 and potential interacting proteins

  • Perform reciprocal Co-IPs to confirm interactions

  • Include appropriate controls (e.g., inactive mutants, unrelated proteins)

• Proximity labeling:

  • Use BioID or APEX2 fusions with RNF185 to identify proximal proteins in living cells

  • Perform mass spectrometry to identify labeled proteins

  • Validate candidates with targeted approaches

• IP-LC-MS/MS workflow:

  • Immunoprecipitate RNF185 complexes from cells under different conditions

  • Identify interacting proteins by mass spectrometry

  • Compare spectral counts to assess enrichment (e.g., for VCP/p97)

• Fluorescence microscopy:

  • Assess colocalization of RNF185 with potential interactors

  • Use fluorescently tagged proteins or specific antibodies

  • Quantify colocalization using appropriate software

• Domain mapping:

  • Generate truncation or point mutants of RNF185 and interacting proteins

  • Determine which domains are essential for interactions

  • This helps understand the structural basis of interactions

These approaches should be combined to build a comprehensive understanding of RNF185's interaction network and how these interactions contribute to its various cellular functions.

How does RNF185 cooperate with other E3 ligases in the ERAD pathway?

RNF185 demonstrates significant functional cooperation with other E3 ligases in the ERAD pathway, particularly with RNF5:

• Functional synergy with RNF5:

  • RNF185 and RNF5 share approximately 70% sequence identity

  • Both target CFTR and CFTRΔF508 for degradation

  • RNF185, like RNF5, controls CFTR stability during translation (co-translational degradation)

  • Combined inactivation of RNF185 and RNF5 leads to dramatic stabilization of CFTRΔF508

• Temporal coordination:

  • RNF185 and RNF5 contribute to co-translational degradation of CFTR

  • Their combined activity is also important for post-translational degradation

  • This suggests a sequential or complementary role throughout the CFTR life cycle

• Integration with other ERAD E3 ligases:

  • Current models suggest RNF5 and GP78 collaborate in CFTRΔF508 degradation

  • RNF5 primes CFTRΔF508 by ubiquitination during translation

  • GP78 subsequently elongates the ubiquitin chain for efficient degradation

  • RNF185 appears to function in parallel with this mechanism

• Protein complex formation:

  • IP-LC-MS/MS studies reveal that RNF170 is a high-confidence interacting protein (HCIP) of both RNF185 and GP78

  • This suggests that ERLIN1/2 interactions could serve as a bridge for larger hetero-oligomeric E3 complexes

  • These complexes may coordinate different aspects of ERAD for specific substrates

• VCP/p97 recruitment:

  • Both RNF185 and other ERAD E3 ligases (e.g., GP78, Hrd1) enrich for VCP/p97

  • VCP/p97 is essential for extracting ubiquitinated proteins from the ER for proteasomal degradation

  • The soluble VCP/p97 cofactors UFD1 and NPLOC4 are also high-confidence interacting proteins of RNF185

This cooperation between multiple E3 ligases likely enables finely tuned regulation of ERAD substrates and provides redundancy in this critical quality control pathway. The RNF5/RNF185 module represents a potential therapeutic target for conditions involving misfolded proteins, such as cystic fibrosis.

How might RNF185 be targeted for therapeutic development?

RNF185 represents a promising therapeutic target across multiple disease contexts, with several potential strategies for intervention:

• Antiviral therapeutics targeting SARS-CoV-2:

  • Small molecules that enhance RNF185-envelope protein interaction could increase viral protein degradation

  • Compounds that increase retention time of the SARS-CoV-2 envelope protein in the ER could enhance its degradation by RNF185

  • The precedent exists for small molecules enhancing E3 ligase-substrate interactions (e.g., β-catenin and SCF β-TrCP)

• Cystic fibrosis treatment approaches:

  • Inhibition of RNF185 (and RNF5) could stabilize CFTRΔF508, potentially restoring some function

  • The RNF5/RNF185 module represents a novel therapeutic target for cystic fibrosis

  • Combined targeting might be more effective than single E3 ligase inhibition

• Cancer metastasis prevention:

  • For prostate cancer, strategies to upregulate or enhance RNF185 activity could limit metastatic potential

  • Alternatively, directly targeting COL3A1 in RNF185-low tumors might prevent enhanced migration and metastasis

  • Patient stratification based on RNF185 expression levels could identify those who would benefit most from such therapies

• Modulation of innate immune responses:

  • Enhancing RNF185 activity could boost antiviral immunity against DNA viruses

  • Inhibiting RNF185 might help control excessive inflammation in autoimmune conditions like SLE where RNF185 is overexpressed

• Drug development approaches:

  • Structure-based design of small molecules targeting the RNF185 RING domain or substrate-binding regions

  • Proteolysis-targeting chimeras (PROTACs) that could either enhance or inhibit RNF185 activity

  • Screening for compounds that modulate specific RNF185-substrate interactions

The therapeutic strategy should be tailored to the specific disease context, as enhancement of RNF185 activity would be beneficial in some conditions (viral infections, cancer metastasis) while inhibition might be preferred in others (cystic fibrosis, certain autoimmune conditions).

What techniques are recommended for investigating RNF185 expression in clinical samples?

For investigating RNF185 expression in clinical samples, researchers should consider the following techniques:

• Quantitative RT-PCR (qRT-PCR):

  • Highly sensitive method for quantifying RNF185 mRNA expression

  • Recommended primers:

    • Human RNF185: 5'-CTGTCACGCCTCTTCCTATTTGT-3' (forward) and 5'-GCCCAGCATTAGGCAATCAG-3' (reverse)

    • Mouse RNF185: 5'-TCTTCTGTTGGCCGTGTTTACA-3' (forward) and 5'-TTGCAGACTGGACACACTTGTC-3' (reverse)

  • Reference genes: GAPDH for cell experiments; 18S RNA and PPIA1 for tissue analyses

• Immunohistochemistry (IHC):

  • Valuable for assessing protein expression and localization in tissue sections

  • Use validated anti-RNF185 antibodies with appropriate controls

  • Consider dual staining with organelle markers (mitochondria, ER) or potential interacting partners

• Western blotting:

  • For quantitative analysis of protein expression

  • Include appropriate loading controls

  • Consider subcellular fractionation to assess localization patterns

• Gene expression profiling:

  • RNA sequencing to assess RNF185 expression in the context of broader transcriptional programs

  • Useful for identifying correlations with disease progression or treatment response

  • Can reveal associated pathways (e.g., EMT in prostate cancer)

• Single-cell analysis:

  • Single-cell RNA sequencing to assess cell-type specific expression

  • Particularly valuable in heterogeneous samples like tumors

  • Can identify specific cell populations with altered RNF185 expression

• Digital spatial profiling:

  • For analyzing RNF185 expression with spatial context in tissue sections

  • Enables correlation with microenvironmental features and other marker expressions

The choice of method should be guided by the specific research question, sample availability, and required sensitivity. For clinical correlation studies, combining multiple approaches provides more robust evidence for RNF185's role in disease mechanisms.

How can researchers design experiments to resolve the dual localization of RNF185 to ER and mitochondria?

Resolving the dual localization of RNF185 to both the ER and mitochondria requires careful experimental design:

• High-resolution imaging approaches:

  • Super-resolution microscopy (STORM, PALM, or SIM) to visualize RNF185 distribution beyond the diffraction limit

  • Live-cell imaging with photoactivatable fluorescent proteins to track movement between organelles

  • Correlative light and electron microscopy (CLEM) to combine fluorescence imaging with ultrastructural analysis

• Specific organelle targeting experiments:

  • Generate chimeric RNF185 constructs with ER or mitochondria-specific targeting sequences

  • Assess whether organelle-restricted RNF185 can perform all functions or only subset-specific roles

  • Use organelle-specific markers (e.g., Tom20 for mitochondrial outer membrane, Sec61 for ER)

• Proximity labeling in specific compartments:

  • Fuse RNF185 with compartment-specific BioID or APEX2 variants

  • Identify proximal proteins in either the ER or mitochondria

  • Compare interactomes to determine compartment-specific functions

• Membrane contact site investigation:

  • Focus on ER-mitochondria contact sites as potential RNF185 enrichment zones

  • Use markers of mitochondria-associated ER membranes (MAMs)

  • Assess the impact of disrupting contact sites on RNF185 localization and function

• Domain mutation analysis:

  • Create precise mutations in the transmembrane domains

  • Identify residues specifically required for ER versus mitochondrial targeting

  • The TM2 domain appears particularly important for proper localization

• Functional rescue experiments:

  • In RNF185-depleted cells, express ER-only, mitochondria-only, or dual-localized RNF185

  • Determine which construct(s) can rescue specific phenotypes

  • This approach helps assign functions to specific subcellular pools of RNF185

• Biochemical fractionation with validation:

  • Perform careful subcellular fractionation to isolate pure ER and mitochondrial fractions

  • Quantify the relative abundance of RNF185 in each compartment

  • Use western blotting with multiple antibodies targeting different epitopes to confirm specificity

These complementary approaches can help resolve whether RNF185 truly has dual localization, whether different pools have distinct functions, and how its distribution might change under various cellular conditions or disease states.

What are the key challenges in studying RNF185 and how might they be addressed?

Studying RNF185 presents several significant challenges that researchers should be aware of:

• Dual localization complexity:

  • RNF185 localizes to both the ER and mitochondria, complicating functional studies

  • Solution: Use compartment-specific targeting constructs and high-resolution imaging techniques to distinguish functions in different locations

• Substrate identification difficulties:

  • E3 ligases often have transient interactions with substrates, making comprehensive identification challenging

  • Solution: Employ substrate trapping approaches using catalytically inactive mutants, proximity labeling techniques, and global ubiquitinome analysis following RNF185 perturbation

• Functional redundancy with RNF5:

  • The high sequence similarity and functional overlap with RNF5 can mask phenotypes in single knockout studies

  • Solution: Generate double knockouts/knockdowns of RNF185 and RNF5, and perform careful epistasis analysis to delineate specific contributions

• Context-dependency of functions:

  • RNF185 appears to have different roles depending on cell type and physiological context

  • Solution: Study RNF185 across multiple cell types, tissue contexts, and disease models to build a comprehensive functional map

• Post-translational regulation:

  • Little is known about how RNF185 itself is regulated

  • Solution: Investigate potential phosphorylation, ubiquitination, or other modifications of RNF185 under different conditions using mass spectrometry and targeted biochemical approaches

• Technical challenges with antibodies:

  • Variable quality of commercial antibodies can complicate detection of endogenous RNF185

  • Solution: Validate antibodies across multiple applications, consider epitope tagging approaches, and use genetic knockouts as negative controls

• Translating basic findings to therapeutic applications:

  • Moving from mechanistic insights to druggable targets presents significant challenges

  • Solution: Focus on protein-protein interaction surfaces that might be amenable to small molecule intervention, and explore approaches to modulate rather than abolish activity

Addressing these challenges requires a multidisciplinary approach combining advanced genetic, biochemical, and imaging techniques. Collaborative efforts between labs with complementary expertise will likely yield the most comprehensive understanding of RNF185 biology.

What are promising future research directions for studying RNF185?

Several promising research directions could significantly advance our understanding of RNF185:

• Comprehensive substrate identification:

  • Apply global proteomics approaches to identify the complete set of RNF185 substrates

  • Use ubiquitin remnant profiling to map specific ubiquitination sites

  • Determine substrate specificity in different cellular compartments and conditions

• Structural biology of RNF185 complexes:

  • Determine the crystal or cryo-EM structure of RNF185 alone and in complex with key substrates

  • Focus on understanding how the RING domain and transmembrane domains contribute to function

  • Use structural insights to guide the design of specific modulators

• Tissue-specific functions in vivo:

  • Generate and characterize tissue-specific conditional knockout mouse models

  • Assess the role of RNF185 in development, homeostasis, and disease progression

  • Determine whether findings from cell culture systems translate to in vivo contexts

• RNF185 in additional viral infections:

  • Expand studies beyond SARS-CoV-2 and HSV-1 to other viral pathogens

  • Determine whether RNF185 represents a broad antiviral mechanism or has virus-specific effects

  • Investigate whether viruses have evolved mechanisms to counteract RNF185-mediated restriction

• Role in additional cancers:

  • Explore whether the tumor-suppressive role observed in prostate cancer extends to other malignancies

  • Identify cancer-specific substrates or regulatory mechanisms

  • Determine whether RNF185 expression correlates with prognosis across cancer types

• Therapeutic targeting approaches:

  • Develop small molecules that can modulate RNF185 activity or specific interactions

  • Explore the potential of RNF185 as a biomarker for disease progression or treatment response

  • Investigate whether RNF185 can be targeted using PROTACs or other emerging therapeutic modalities

• Integration with cellular stress responses:

  • Explore how RNF185 contributes to integrated cellular responses to ER stress, mitochondrial dysfunction, and innate immune activation

  • Determine whether RNF185 functions as a node connecting these distinct stress pathways

  • Investigate potential roles in cellular adaptation and resilience

These research directions would provide a more comprehensive understanding of RNF185 biology and could lead to novel therapeutic approaches for multiple diseases.

How might researchers resolve contradictions in the RNF185 literature?

Several apparent contradictions exist in the RNF185 literature. Here are strategies to resolve these inconsistencies:

• Localization discrepancies (ER vs. mitochondria):

  • Contradiction: Some studies report RNF185 as primarily an ER protein involved in ERAD , while others describe it as a mitochondrial protein regulating mitophagy .

  • Resolution approach:

    • Quantify relative distribution across compartments using biochemical fractionation with stringent controls

    • Investigate whether distribution changes under different cellular conditions or stress responses

    • Consider the possibility that different splice variants or post-translationally modified forms might have distinct localizations

• Functional roles in autophagy vs. ERAD:

  • Contradiction: RNF185 has been implicated in both selective mitochondrial autophagy and ER-associated degradation .

  • Resolution approach:

    • Determine whether these represent truly distinct functions or different aspects of a broader quality control role

    • Investigate potential condition-specific roles (e.g., different functions under basal vs. stress conditions)

    • Examine whether different protein complexes mediate these distinct functions

• Different ubiquitin chain specificities:

  • Contradiction: RNF185 has been reported to catalyze K27-linked chains (on cGAS) , K63-linked chains (on BNIP1) , and presumably K48-linked chains (for proteasomal degradation of CFTR) .

  • Resolution approach:

    • Directly compare ubiquitination patterns on different substrates under identical conditions

    • Investigate whether RNF185 has intrinsic chain-type specificity or whether this is determined by cofactors or substrates

    • Examine whether different E2 enzymes pair with RNF185 for different substrates (e.g., UBE2J1/UBE2J2 for ERAD)

• Divergent phenotypic effects:

  • Contradiction: RNF185 appears to have both pro-survival (protecting from ER stress) and anti-growth (limiting cancer progression) effects in different contexts.

  • Resolution approach:

    • Carefully define the cellular context and experimental conditions when comparing studies

    • Investigate cell type-specific effects and signaling pathway interactions

    • Consider the possibility that the net effect of RNF185 depends on the balance between different substrates in specific contexts

• Methodological approaches:

  • General strategy:

    • Reproduce key experiments using multiple cell lines and primary cells

    • Employ complementary techniques (genetic, biochemical, imaging) to validate findings

    • Use both gain- and loss-of-function approaches with appropriate controls

    • Consider the impact of overexpression artifacts versus physiological expression levels

By systematically addressing these contradictions with rigorous experimental design and attention to context-specific effects, researchers can develop a more unified understanding of RNF185's multifaceted roles in cellular biology.