Recombinant Rat E3 ubiquitin-protein ligase RNF185 (Rnf185)

What is RNF185 and what is its primary cellular function?

RNF185 is a RING finger domain-containing E3 ubiquitin-protein ligase that plays multiple roles in cellular homeostasis. Its primary function involves catalyzing the transfer of ubiquitin to specific substrate proteins, marking them for degradation or altering their function. RNF185 has been characterized as a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy (mitophagy) in cultured cells . Additionally, it functions in ER-associated protein degradation (ERAD), participating in quality control of proteins synthesized on the ER membrane . The protein contains two C-terminal transmembrane domains that mediate its localization to the mitochondrial outer membrane .

Where is RNF185 primarily localized in cells?

RNF185 exhibits dual localization within cells. Studies using confocal microscopy with DsRed2-Mito and GFP-RNF185 have demonstrated significant colocalization at mitochondria in HeLa cells . Endogenous RNF185 labeled with Alexa Fluor 488 has been shown to completely overlap with MitoTracker Red, confirming its mitochondrial localization . This localization has been further substantiated through differential centrifugation techniques, which show RNF185 to be most abundant in mitochondria-enriched fractions .

More recent studies have also identified RNF185 as an ER-resident ubiquitin ligase that participates in the ERAD complex . This dual localization suggests that RNF185 may perform specialized functions depending on its subcellular context, making it an important regulator of organelle homeostasis.

How is the RING domain of RNF185 involved in its ubiquitin ligase activity?

The RING domain of RNF185 is essential for its E3 ubiquitin ligase activity. Experimental evidence shows that mutations in the RING domain (RNF185-RM) significantly decrease the protein's polyubiquitination capability compared to wild-type RNF185 . This indicates that the structural integrity of the RING domain is critical for RNF185's ability to transfer ubiquitin to substrate proteins.

Interestingly, deletion of the transmembrane domains (RNF185-TM) almost completely abolishes RNF185's self-polyubiquitination activity, suggesting that proper subcellular localization is equally crucial for its function as an E3 ligase . Both the RING domain and transmembrane domains are therefore required for RNF185 to function properly in the ubiquitination pathway.

How does RNF185 regulate mitochondrial autophagy?

RNF185 regulates mitochondrial autophagy through a sophisticated mechanism involving LC3 and selective substrate ubiquitination. When overexpressed, RNF185 stimulates LC3II accumulation and autophagolysosome formation in human cell lines . The process depends on RNF185's intact RING domain and transmembrane domains, as demonstrated by experiments with mutated forms of RNF185.

Mechanistically, RNF185 has been shown to mediate K63-linked polyubiquitination of substrates like BNIP1, a Bcl-2 family protein . This polyubiquitinated BNIP1 subsequently recruits the autophagy receptor p62, which acts as a bridge by binding both ubiquitin and LC3, thereby linking ubiquitination to autophagy machinery . This process explains how RNF185 facilitates the selective degradation of mitochondria through the autophagy pathway.

When RNF185 is overexpressed in cells, they display reduced reactive oxygen species (ROS) levels despite showing abnormal morphology with globular, shrinking, and punctate cell shapes . This reduction in ROS is consistent with the loss of mitochondrial mass through autophagy, as mitochondria are major sources of cellular ROS.

What are the known protein interactions and substrates of RNF185?

RNF185 interacts with several key proteins as part of its cellular functions:

• BNIP1 (Bcl-2 Nineteen kilodalton Interacting Protein 1): Co-immunoprecipitation experiments have demonstrated that RNF185 strongly binds to BNIP1 . This interaction depends on their transmembrane domains rather than the RING domain . RNF185 catalyzes K63-linked polyubiquitination of BNIP1, which then recruits autophagy machinery.

• ATG5: RNF185 also interacts with ATG5, an essential component of autophagy, although this binding appears weaker than its interaction with BNIP1 .

• cGAS (cyclic GMP-AMP synthase): During HSV-1 infection, RNF185 interacts with cGAS and catalyzes its K27-linked polyubiquitination, which promotes cGAS enzymatic activity in innate immune responses .

• SARS-CoV-2 envelope protein: RNF185 colocalizes with the SARS-CoV-2 envelope protein at the ER and regulates its degradation . This interaction has functional consequences for viral production, as depletion of RNF185 increases viral titers.

• TMEM259: This protein is necessary for RNF185 function in the context of the ERAD complex for quality control of membrane proteins .

How is RNF185 implicated in disease pathogenesis?

RNF185 plays significant roles in multiple disease contexts:

• Viral Infections: RNF185 regulates SARS-CoV-2 envelope protein stability, with its depletion resulting in increased viral titers for multiple SARS-CoV-2 strains . It also facilitates cGAS-mediated innate immune responses during HSV-1 infection, suggesting a broader role in antiviral immunity .

• Cancer Progression: Patient data analysis reveals a negative correlation between RNF185 expression and prostate cancer progression and metastasis . RNF185 depletion in prostate cancer cell lines enhances migration and invasion capabilities, and in mouse models, it results in larger tumors and more frequent lung metastases .

• Autoimmune Diseases: Patients with Systemic Lupus Erythematosus (SLE) display elevated expression of RNF185 mRNA, suggesting its potential involvement in autoimmune pathogenesis .

These diverse disease associations highlight the multifaceted roles of RNF185 in cellular homeostasis and pathology.

How does RNF185 contribute to innate immune signaling pathways?

RNF185 serves as a positive regulator of the cGAS-STING signaling pathway, which is crucial for cytosolic DNA sensing and innate immune responses. During viral infections like HSV-1, RNF185 interacts with cGAS and catalyzes its K27-linked polyubiquitination . This specific ubiquitination enhances cGAS enzymatic activity rather than targeting it for degradation.

Functionally, ectopic expression of RNF185 enhances IRF3-responsive gene expression, while its knockdown impairs these responses . This modulation of cGAS activity by RNF185 represents a novel regulatory mechanism in innate immunity. The significance of this pathway is underscored by observations in Systemic Lupus Erythematosus (SLE) patients, who display elevated expression of RNF185 mRNA .

The identification of RNF185 as the first E3 ubiquitin ligase for cGAS provides important insights into how cGAS activity is dynamically regulated during immune responses. This mechanism may represent a potential therapeutic target for both infectious diseases and autoimmune conditions.

What is the relationship between RNF185 and the SARS-CoV-2 viral life cycle?

RNF185 plays a critical role in regulating SARS-CoV-2 infection by controlling the stability of the viral envelope protein. The envelope protein is synthesized in the ER and subsequently trafficked to the Golgi and ER-Golgi intermediate compartment, where it participates in viral assembly, budding, viral release, and inflammasome activation .

Experimental evidence shows that RNF185 and the SARS-CoV-2 envelope protein partially co-localize with ER markers . When RNF185 is knocked out using CRISPR-Cas9, levels of SARS-CoV-2 envelope protein increase by 2- to 3-fold across multiple cell lines . This regulation extends to envelope proteins from various SARS-CoV-2 clinical variants (including those with mutations in the C-terminus or transmembrane domain) and SARS-CoV, but not to the more divergent MERS envelope protein .

The biological significance of this interaction is demonstrated by viral titer assays, where depletion of RNF185 in A549-ACE2 cells results in approximately 60% increase in viral titers for multiple SARS-CoV-2 strains . This finding indicates that RNF185-mediated regulation of the envelope protein has direct consequences for virus production and could potentially be targeted for antiviral therapy development.

How does RNF185 regulate cancer progression through COL3A1?

RNF185 functions as a gatekeeper of prostate cancer metastasis partly through its control of COL3A1 (collagen type III alpha 1 chain) expression. Analysis of patient data has revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis . This clinical observation is supported by experimental evidence showing that RNF185 depletion enhances the migration and invasion capabilities of prostate cancer cell lines .

Mechanistically, RNA-sequencing and Ingenuity Pathway Analysis of RNF185-depleted prostate cancer cells identified upregulation of wound-healing and cellular movement pathways . Gene Set Enrichment Analyses confirmed the deregulation of genes implicated in epithelial-to-mesenchymal transition, with COL3A1 identified as the primary mediator of RNF185's impact on migration phenotypes .

The functional relationship between RNF185 and COL3A1 is further validated by experiments showing that the enhanced migration and metastasis of RNF185 knockdown prostate cancer cells can be attenuated by co-inhibition of COL3A1 . This suggests that both RNF185 and COL3A1 may serve as novel markers and potential therapeutic targets for prostate tumors.

What techniques are most effective for studying RNF185 subcellular localization?

Several complementary techniques have proven effective for accurately determining RNF185 subcellular localization:

• Confocal Microscopy with Fluorescent Fusion Proteins: Co-expression of DsRed2-Mito (a mitochondrial marker) with GFP-RNF185 has successfully demonstrated mitochondrial colocalization . Similarly, RNF185-iRFP720 fusion constructs can be used alongside organelle-specific dyes to visualize localization to the ER or other compartments .

• Immunofluorescence with Organelle Markers: Using affinity-purified anti-RNF185 polyclonal antibodies combined with MitoTracker Red or other organelle-specific dyes provides validation of endogenous protein localization . This approach avoids potential artifacts from overexpression of tagged proteins.

• Differential Centrifugation: Biochemical fractionation through differential centrifugation offers quantitative assessment of RNF185 distribution across cellular compartments . This technique complements imaging approaches by providing a population-level view of protein distribution.

• Deletion Analysis with Transmembrane Domain Mutants: Expressing RNF185 constructs with deleted or mutated transmembrane domains helps determine the specific sequences required for proper localization . These experiments are essential for understanding the structural basis of RNF185 targeting to different organelles.

For optimal results, researchers should employ at least two independent methods to confirm RNF185 localization, preferably combining imaging and biochemical approaches.

How can researchers effectively assess RNF185 E3 ligase activity?

Assessment of RNF185 E3 ligase activity can be accomplished through several well-established methods:

• Self-Polyubiquitination Assays: RNF185 undergoes intensive self-polyubiquitination, which can be detected by co-expressing tagged RNF185 with tagged ubiquitin, followed by immunoprecipitation and Western blotting . This serves as a useful proxy for E3 ligase activity and can be used to compare wild-type RNF185 with RING domain mutants.

• Substrate Ubiquitination Assays: For known substrates like BNIP1 or cGAS, co-expressing the substrate with RNF185 and ubiquitin, followed by immunoprecipitation of the substrate and detection of ubiquitin chains, provides direct evidence of E3 ligase activity toward specific targets .

• Ubiquitin Linkage-Specific Antibodies: RNF185 has been shown to catalyze K63-linked polyubiquitination of BNIP1 and K27-linked polyubiquitination of cGAS . Using linkage-specific antibodies or ubiquitin mutants can help determine the type of ubiquitin chains formed.

• In Vitro Ubiquitination Assays: Reconstituting the ubiquitination reaction with purified components (E1, E2, RNF185, substrate, ubiquitin, and ATP) provides the most direct assessment of E3 ligase activity under controlled conditions.

• Functional Readouts: For RNF185's role in autophagy, monitoring LC3I to LC3II conversion and GFP-LC3 puncta formation in the presence of wild-type or mutant RNF185 offers a functional assessment of its activity .

When evaluating RNF185 activity, it is essential to include appropriate controls such as RING domain mutants (RNF185-RM) and transmembrane domain deletion mutants (RNF185-TM), as both domains are critical for full activity .

What are the optimal approaches for manipulating RNF185 expression in experimental models?

Several effective strategies exist for modulating RNF185 expression in experimental systems:

• CRISPR-Cas9 Knockout: CRISPR-Cas9 has been successfully used to deplete RNF185 in multiple cell lines (HEK293T, K562, A549) with 2-4 sgRNAs targeting different regions of the gene . This approach provides stable and complete loss of RNF185 expression, as validated by Western blotting.

• RNA Interference: Short hairpin RNA (shRNA) against RNF185 has been effectively employed in prostate cancer cell lines and mouse models to study its role in cancer progression . This approach may be preferable when partial knockdown is desired or when complete knockout causes severe phenotypes.

• Transient Overexpression: Transfection of expression constructs for wild-type or mutant forms of RNF185 (with appropriate epitope tags such as Flag, GFP, or RFP) provides a means to study gain-of-function effects and structure-function relationships .

• Stable Cell Lines: For long-term studies, establishing stable cell lines with inducible RNF185 expression using systems like Tet-On/Off can help mitigate potential toxicity issues associated with constitutive overexpression.

• Animal Models: For in vivo studies, subcutaneous inoculation of cells with RNF185 knockdown has been used to assess effects on tumor growth and metastasis in mice . This approach can be extended to create transgenic or knockout mouse models for more comprehensive in vivo analyses.

When manipulating RNF185 expression, researchers should consider potential compensatory mechanisms involving other E3 ligases and validate knockdown or overexpression at both RNA and protein levels. Additionally, including appropriate control constructs (empty vectors, non-targeting sgRNAs, or inactive mutants) is essential for rigorous experimental design.

How might RNF185 be targeted for therapeutic development?

Given its roles in viral infection, cancer progression, and immune regulation, RNF185 represents a promising therapeutic target with multiple potential applications:

• Antiviral Therapies: Since depletion of RNF185 increases SARS-CoV-2 viral titers, enhancing its activity might suppress viral replication . Conversely, for viruses that might exploit RNF185 for their life cycle, inhibitors could be beneficial. Developing small molecules that modulate RNF185's interaction with viral proteins or alter its E3 ligase activity could provide novel antiviral strategies.

• Cancer Therapeutics: For prostate cancer, where RNF185 functions as a metastasis suppressor, approaches to restore or enhance its expression or activity could potentially limit cancer progression . This might involve gene therapy approaches or small molecules that mimic RNF185's effects on downstream targets like COL3A1.

• Autoimmune Disease Management: Given the elevated expression of RNF185 in SLE patients and its role in innate immune signaling, inhibiting its activity might help manage overactive immune responses in autoimmune conditions .

Therapeutic development would likely focus on one of three approaches: (1) directly modulating RNF185 expression levels, (2) altering its E3 ligase activity with small molecules, or (3) targeting specific RNF185-substrate interactions. Each approach would require extensive validation in disease-relevant models before clinical translation.

What are the unexplored functions of RNF185 in cellular homeostasis?

Despite significant advances in understanding RNF185, several aspects of its biology remain unexplored:

• Regulation of RNF185 Expression: Little is known about how RNF185 expression is regulated under different physiological and pathological conditions. Identifying transcription factors and epigenetic mechanisms controlling RNF185 expression could reveal additional regulatory layers.

• Post-translational Modifications: Whether RNF185 itself undergoes regulatory post-translational modifications beyond self-ubiquitination remains largely unexplored. Phosphorylation, SUMOylation, or other modifications might fine-tune its activity or localization.

• Substrate Recognition Mechanisms: The structural basis for RNF185's specificity toward different substrates (BNIP1, cGAS, SARS-CoV-2 envelope) is not fully understood. Detailed structural studies could illuminate the principles of substrate recognition.

• Interactions with Other E3 Ligases: Potential cooperation or competition between RNF185 and other E3 ligases targeting similar substrates or pathways represents an interesting area for investigation.

• Roles in Development and Aging: The physiological functions of RNF185 during development, differentiation, and aging remain largely unexplored, particularly in complex organisms.

Investigation of these unexplored aspects would provide a more comprehensive understanding of RNF185's roles in cellular homeostasis and disease.

What are common challenges in working with recombinant RNF185 and how can they be addressed?

Researchers working with recombinant RNF185 often encounter several technical challenges:

• Protein Solubility and Purification: As a transmembrane protein, RNF185 can be difficult to express and purify in its full-length form. Solutions include:

  • Using detergent-based extraction methods optimized for membrane proteins

  • Creating soluble truncated versions lacking the transmembrane domains for certain applications

  • Expressing the protein with solubility-enhancing tags such as MBP or SUMO

• Maintaining Enzymatic Activity: E3 ligases often lose activity during purification processes. Strategies to preserve activity include:

  • Minimizing freeze-thaw cycles

  • Including reducing agents like DTT to maintain the integrity of the RING domain

  • Conducting activity assays immediately after purification

• Subcellular Targeting: When expressing recombinant RNF185 in cells, proper localization to mitochondria or ER is crucial for function. Approaches include:

  • Verifying localization with fluorescent tags or organelle markers

  • Ensuring the transmembrane domains are intact and properly oriented

  • Using cell type-specific optimized expression systems

• Distinguishing Substrate Specificity: Determining which proteins are bona fide RNF185 substrates versus interacting partners can be challenging. Methods to address this include:

  • Using RING domain mutants as negative controls for ubiquitination

  • Performing in vitro ubiquitination assays with purified components

  • Validating interactions through multiple independent techniques

By anticipating these challenges and implementing appropriate technical solutions, researchers can enhance the reliability and reproducibility of their work with recombinant RNF185.

How can researchers differentiate between direct and indirect effects of RNF185 in experimental systems?

Distinguishing direct from indirect effects of RNF185 requires careful experimental design:

• Structure-Function Analysis: Comparing wild-type RNF185 with catalytically inactive mutants (RING domain mutants) can help determine whether observed effects depend on ubiquitination activity or protein-protein interactions .

• Acute vs. Chronic Manipulation: Using inducible systems for RNF185 expression or depletion allows examination of immediate effects before compensatory mechanisms engage, helping identify direct consequences of RNF185 activity.

• In Vitro Reconstitution: For ubiquitination events, reconstituting the reaction with purified components provides direct evidence of RNF185's activity toward specific substrates without cellular confounding factors.

• Rescue Experiments: Restoring wild-type RNF185 expression in knockout cells should reverse phenotypes directly caused by RNF185 deficiency, while indirect effects might persist or respond differently.

• Domain Interaction Mapping: Identifying specific domains or residues of RNF185 that mediate interaction with different partners can help dissect pathway-specific functions. For instance, the transmembrane domains mediate BNIP1 binding, while other regions might be involved in cGAS interaction .

• Temporal Analysis: Monitoring the kinetics of molecular changes following RNF185 manipulation can help establish cause-effect relationships, with direct effects typically occurring more rapidly than indirect consequences.

Implementing these approaches in combination provides stronger evidence for distinguishing direct RNF185 functions from downstream or compensatory effects in complex biological systems.

What statistical approaches are most appropriate for analyzing RNF185 expression data in patient samples?

When analyzing RNF185 expression data from patient samples, several statistical approaches are particularly valuable:

• Correlation Analysis: For examining relationships between RNF185 expression and clinical parameters (e.g., tumor stage, metastasis, survival), Pearson or Spearman correlation coefficients are appropriate depending on data distribution. This approach has successfully identified negative correlations between RNF185 expression and prostate cancer progression .

• Survival Analysis: Kaplan-Meier curves with log-rank tests can reveal associations between RNF185 expression levels and patient outcomes. Cox proportional hazards regression enables multivariate analysis to determine whether RNF185 is an independent prognostic factor.

• Gene Set Enrichment Analysis (GSEA): This approach has been effectively used to identify biological pathways associated with low RNF185 expression in patient samples, such as epithelial-to-mesenchymal transition genes in prostate cancer .

• Receiver Operating Characteristic (ROC) Analysis: To evaluate RNF185's potential as a diagnostic or prognostic biomarker, ROC analysis can determine optimal expression thresholds and assess sensitivity/specificity.

• Paired Sample Analysis: For comparing RNF185 expression between tumor and adjacent normal tissue from the same patient, paired t-tests or Wilcoxon signed-rank tests provide greater statistical power by controlling for inter-individual variation.

When applying these methods, researchers should address potential confounding factors through appropriate multivariate models and validate findings in independent cohorts whenever possible.

How should researchers interpret changes in RNF185 activity across different experimental conditions?

Interpreting changes in RNF185 activity requires consideration of multiple factors: