Recombinant Mouse E3 ubiquitin-protein ligase RNF185 (Rnf185)

What is RNF185 and what is its basic structure?

RNF185 is a RING finger domain-containing E3 ubiquitin ligase primarily localized to the endoplasmic reticulum (ER). The protein contains a catalytic RING domain essential for its ubiquitin ligase activity, and its functional domains include the RING finger motif (containing critical cysteine residues at positions 39 and 42) and transmembrane domains that anchor it to the ER membrane. In experimental settings, researchers have generated various mutants including RNF185 ΔC (amino acids 1-176), RNF185 ΔR (amino acids 94-192), and RNF185 RING mutant (C39A, C42A) to study domain-specific functions .

How is RNF185 related to other E3 ligases?

RNF185 is a structural and functional homolog of RNF5/RMA1. These two E3 ligases share sequence similarity and can function cooperatively in certain cellular contexts. While they have some overlapping substrates and functions, they also demonstrate distinct substrate specificities. For instance, both RNF185 and RNF5 target CFTR for degradation, but they can act both independently and synergistically depending on the cellular context and translation status of the substrate .

What are the known physiological functions of RNF185?

RNF185 has several documented physiological functions:

• Regulation of ER-associated protein degradation (ERAD)

• Quality control of transmembrane proteins like CFTR

• Modulation of innate immune responses through cGAS ubiquitination

• Regulation of cancer cell migration and metastasis through control of COL3A1 expression

• Viral defense mechanisms, including regulation of SARS-CoV-2 envelope protein stability

Each function involves specific protein interactions and ubiquitin-mediated regulatory mechanisms .

How does RNF185 contribute to ERAD?

RNF185 functions as a key E3 ligase in the ER-associated degradation (ERAD) pathway, specifically targeting misfolded or improperly assembled transmembrane proteins. It recognizes substrate proteins during or shortly after their translation and catalyzes their ubiquitination, marking them for proteasomal degradation.

RNF185 works in coordination with other components of the ERAD machinery, including chaperones that identify misfolded proteins and the proteasome that ultimately degrades ubiquitinated substrates. Importantly, RNF185 can function both during protein translation (co-translational degradation) and after synthesis is complete (post-translational degradation), providing a comprehensive quality control mechanism .

What is the relationship between RNF185 and CFTR degradation?

RNF185 specifically targets the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant CFTRΔF508 for degradation through the proteasome pathway. This activity is dependent on RNF185's RING domain and proteasome function.

The mechanism involves:

• Recognition of CFTR/CFTRΔF508 during translation

• Ubiquitination of the target protein

• Delivery to the proteasome for degradation

Importantly, RNF185 works in conjunction with RNF5 to control CFTR stability. While RNF185 alone can target CFTR for co-translational degradation, the combined action of RNF185 and RNF5 is required for efficient post-translational degradation of CFTR, particularly the CFTRΔF508 mutant. This represents a potential therapeutic target for cystic fibrosis treatment .

Does RNF185 show substrate specificity in ERAD?

Yes, RNF185 demonstrates clear substrate specificity in ERAD. Despite being an ER-associated E3 ligase, RNF185 does not indiscriminately target all ERAD substrates. Research has shown that while RNF185 controls the stability of CFTR and CFTRΔF508, it does not affect the degradation of other classical ERAD model substrates such as CD3δ, TCRα, or α1-antitrypsin mutants (NHK, Z mutants).

This substrate specificity suggests that RNF185 recognizes specific structural features or protein interactions unique to its targets, rather than acting as a general quality control factor for all misfolded ER proteins .

How does RNF185 modulate innate immune responses?

RNF185 serves as a positive regulator of DNA-sensing innate immune pathways by targeting cyclic GMP-AMP synthase (cGAS). Upon viral DNA stimulation, such as during HSV-1 infection, RNF185 interacts with cGAS and catalyzes its K27-linked polyubiquitination. This modification doesn't target cGAS for degradation but instead enhances its enzymatic activity.

Enhanced cGAS activity leads to increased production of the second messenger 2'3'-cGAMP, which binds to STING (stimulator of interferon genes) and activates downstream signaling, ultimately resulting in stronger IRF3-responsive gene expression and antiviral responses .

What is the mechanism of RNF185-mediated cGAS regulation?

The mechanism involves:

• RNF185 interacts with cGAS during viral infection (e.g., HSV-1)

• RNF185 specifically catalyzes K27-linked polyubiquitination of cGAS (not K48-linked, which would lead to degradation)

• This K27-linked ubiquitination promotes cGAS enzymatic activity

• Enhanced cGAS activity increases 2'3'-cGAMP production

• 2'3'-cGAMP activates STING-dependent signaling

• This leads to enhanced IRF3 activation and antiviral gene expression

Importantly, knockdown of RNF185 significantly attenuates IRF3-responsive gene expression during viral infection, confirming its role as a positive regulator of this pathway .

Is RNF185 implicated in autoimmune diseases?

Yes, RNF185 has been implicated in autoimmune disorders, particularly Systemic Lupus Erythematosus (SLE). Studies have shown that SLE patients display elevated expression of RNF185 mRNA compared to healthy controls. This elevation is consistent with RNF185's role in enhancing cGAS activity, as dysregulated cGAS-STING signaling has been linked to autoimmune pathogenesis.

The abnormal activation of cytosolic DNA sensing pathways by self-DNA is a key feature of several autoimmune diseases. By enhancing cGAS activity through ubiquitination, elevated RNF185 levels may contribute to the hyperactive immune responses observed in SLE patients .

What is the relationship between RNF185 and cancer progression?

RNF185 appears to function as a tumor suppressor in certain contexts, particularly in prostate cancer. Analysis of patient data has revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis.

Lower RNF185 expression is associated with:

• More advanced disease stages

• Increased metastatic potential

• Poorer patient outcomes

This relationship has been experimentally validated using cell lines and mouse models, where RNF185 depletion resulted in enhanced migration and invasion capabilities of prostate cancer cells in culture, larger tumors, and more frequent lung metastases in mice .

How does RNF185 regulate cancer cell migration and metastasis?

RNF185 regulates cancer cell migration and metastasis primarily through its control of COL3A1 (collagen type III alpha 1 chain) expression. RNA-sequencing and pathway analyses identified wound-healing and cellular movement among the most significant pathways upregulated in RNF185-depleted prostate cancer cells.

The mechanism involves:

• RNF185 normally suppresses COL3A1 levels

• When RNF185 is depleted, COL3A1 expression increases

• Elevated COL3A1 promotes epithelial-to-mesenchymal transition (EMT)

• EMT enhances cancer cell migration and invasion

• This ultimately leads to increased metastatic potential

Supporting this mechanism, enhanced migration and metastasis of RNF185 knockdown prostate cancer cells were attenuated upon co-inhibition of COL3A1, confirming COL3A1 as the primary mediator of these phenotypes .

Can RNF185 serve as a prognostic marker in cancer?

Based on current research, RNF185 shows potential as a prognostic marker, particularly in prostate cancer. The negative correlation between RNF185 expression and cancer progression suggests that low RNF185 levels may predict more aggressive disease and poorer outcomes.

Additionally, the relationship between RNF185 and COL3A1 provides a mechanistic basis for its prognostic value. Both RNF185 and COL3A1 could potentially serve as novel markers for assessing the metastatic potential of prostate tumors, helping to identify patients who might benefit from more aggressive treatment approaches .

How does RNF185 interact with viral proteins?

RNF185 has been identified as a regulator of viral protein stability, particularly for the SARS-CoV-2 envelope protein. As an ER-resident E3 ubiquitin ligase, RNF185 co-localizes with the SARS-CoV-2 envelope protein at the endoplasmic reticulum, where it can regulate the stability of this viral protein through the ubiquitin-proteasome pathway.

This interaction represents a potential host defense mechanism against viral infection, as the degradation of viral structural proteins could interfere with viral assembly and propagation .

What is the impact of RNF185 on SARS-CoV-2 infection?

RNF185 appears to function as a restrictive factor in SARS-CoV-2 infection. Experimental evidence shows that depletion of RNF185 significantly increases SARS-CoV-2 viral titer in cellular models, suggesting that RNF185 normally limits viral replication.

The mechanism likely involves:

• RNF185 targeting the SARS-CoV-2 envelope protein for degradation

• Reduced envelope protein availability

• Impaired viral assembly and replication

• Lower viral titers and reduced infection

This finding has important implications for understanding host-virus interactions and may provide insights for developing novel antiviral therapeutic strategies .

Could targeting RNF185 be a therapeutic strategy for viral infections?

Modulation of RNF185 activity could potentially serve as a novel therapeutic approach for viral infections, though with important considerations:

For enhancing RNF185 activity:

• Could increase degradation of viral proteins

• May limit viral replication and spread

• Could enhance innate immune responses through cGAS pathway

For inhibiting RNF185 activity:

• May be beneficial in contexts where excessive immune activation is harmful

• Could reduce autoimmune complications in severe viral infections

The optimal approach would depend on the specific viral infection, disease stage, and patient factors. Further research is needed to determine the therapeutic window and potential side effects of RNF185 modulation in viral infections .

What are the established methods for studying RNF185 ubiquitination activity?

In vitro ubiquitination assay:

• Express and purify GST-tagged RNF185 (wild-type or mutant) from bacteria using FPLC with fast-flow GST columns

• Set up reaction mixtures containing:

  • 0.5 M HEPES, pH 8.0

  • 250 nM E1 enzyme

  • 600 μM ubiquitin

  • 1 mM Mg-ATP

  • 0.4 μM of appropriate E2 enzymes

  • Purified RNF185

• Incubate at 37°C

• Terminate reactions with SDS sample buffer

• Separate proteins by SDS-PAGE

• Visualize by immunoblot using anti-GST and anti-ubiquitin antibodies

This approach allows for direct assessment of RNF185's E3 ligase activity and can help identify compatible E2 enzymes and specific ubiquitin linkage types .

What methods are used to detect RNF185-substrate interactions?

Co-immunoprecipitation (Co-IP):

• Lyse cells in appropriate buffer (e.g., 50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, with protease inhibitors)

• For CFTR/RNF185 interactions specifically, use 20 mM HEPES, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40

• Preclear lysates with Sepharose 4B beads

• Immunoprecipitate with specific antibodies

• Wash extensively

• Analyze by SDS-PAGE and immunoblotting

For endogenous RNF185:
Monitor levels by immunoprecipitation using RNF185-specific antibodies followed by immunoblotting, as direct detection can be challenging due to low expression levels .

What genetic tools have been developed to manipulate RNF185 expression?

Several genetic tools have been developed for RNF185 research:

For overexpression:

• pcDNA3.1 FLAG-RNF185 vectors expressing full-length protein

• Domain deletion mutants: RNF185 ΔC (1-176), RNF185 ΔR (94-192)

• RING mutant (C39A, C42A) that lacks E3 ligase activity

For knockdown:

• siRNA oligonucleotides targeting RNF185 (sequences: 5′-GAUAUUUGCCACAGCAUUU-3′ or 5′-CUUCUGUUGGCCGUGUUUA-3′)

• shRNA constructs for stable knockdown expressed in pSS-H1 vector

For knockout:

• sgRNAs targeting RNF185 for CRISPR/Cas9-mediated gene editing

These tools enable functional studies of RNF185 through gain-of-function and loss-of-function approaches .

How do RNF185 and RNF5 cooperate in substrate regulation?

RNF185 and RNF5 demonstrate a complex functional relationship with both distinct and overlapping roles. While both target CFTR for degradation, they show different temporal patterns of activity and can synergize for more efficient substrate processing.

Temporal cooperation:

• RNF5 and RNF185 can independently target CFTR for co-translational degradation

• Combined action of both E3 ligases is required for efficient post-translational degradation

• Simultaneous depletion profoundly blocks CFTRΔF508 degradation both during and after translation

Mechanistic model:

• RNF5 may prime CFTRΔF508 by initial ubiquitination during translation

• RNF185 may then contribute to this process

• Together they facilitate efficient degradation throughout the protein's lifecycle

This cooperative activity represents a novel E3 ligase module specifically tailored to CFTR quality control, with potential implications for therapeutic development in cystic fibrosis .

What is known about tissue-specific expression patterns of RNF185?

RNF185 shows differential expression across tissues, though comprehensive tissue profiling has not been fully reported. Research has examined RNF185 expression in various contexts:

Methodological approach:

• RT-qPCR analysis using transcript-specific primers

• For mouse tissues: mouse RNF185-specific primers (5′-TCTTCTGTTGGCCGTGTTTACA-3′ forward and 5′-TTGCAGACTGGACACACTTGTC-3′ reverse)

• For human tissues: human RNF185-specific primers (5′-CTGTCACGCCTCTTCCTATTTGT-3′ forward and 5′-GCCCAGCATTAGGCAATCAG-3′ reverse)

• Using appropriate reference genes (18S RNA and PPIA1 for tissue analysis)

Understanding tissue-specific expression patterns is crucial for predicting potential side effects of therapeutic interventions targeting RNF185 and for understanding its physiological roles in different organ systems .

How is RNF185 expression regulated during stress conditions?

RNF185 expression can be modulated by cellular stress conditions, particularly those affecting the endoplasmic reticulum. Studies have examined how RNF185 levels change during the unfolded protein response (UPR):

Experimental approach:

• Treat cells with UPR inducers such as tunicamycin (2 μg/ml)

• Harvest cells at different time points

• Evaluate RNF185 expression by qPCR

• Use GRP78 as a control for UPR induction

• Normalize to appropriate housekeeping genes (e.g., GAPDH)

Understanding how RNF185 responds to cellular stress provides insights into its role in adaptive responses and potential implications for diseases associated with ER stress, such as neurodegenerative disorders, diabetes, and cancer .

What are the potential therapeutic applications of targeting RNF185?

Based on current research, several therapeutic applications for targeting RNF185 show promise:

For Cystic Fibrosis:

• Inhibiting RNF185/RNF5 to reduce degradation of CFTRΔF508

• Potentially allowing more mutant CFTR to reach the cell surface

• Combining with CFTR correctors for enhanced therapeutic effect

For Cancer:

• Upregulating or restoring RNF185 expression in prostate cancer

• Targeting the RNF185-COL3A1 axis to reduce metastatic potential

• Using RNF185 status as a biomarker for patient stratification

For Viral Infections:

• Enhancing RNF185 activity to increase viral protein degradation

• Developing molecules that mimic or boost RNF185's antiviral functions

For Autoimmune Diseases:

• Inhibiting RNF185 to reduce excessive cGAS-STING activation

• Potentially alleviating symptoms in conditions like SLE

Each application requires further research to validate targets and develop specific modulators of RNF185 activity .

What are the current gaps in RNF185 research?

Despite significant progress, several knowledge gaps remain in RNF185 research:

Structural biology:

• Detailed three-dimensional structure of RNF185

• Structural basis for substrate recognition and specificity

• Interaction sites with E2 enzymes and substrates

Physiological functions:

• Complete tissue expression profile and physiological roles

• Phenotypic consequences of RNF185 knockout in animal models

• Role in development and tissue homeostasis

Regulatory mechanisms:

• Comprehensive understanding of RNF185 transcriptional and post-translational regulation

• Factors that modulate its E3 ligase activity

• Potential auto-regulatory mechanisms

Disease associations:

• Comprehensive assessment of RNF185 alterations across disease states

• Genetic variations affecting RNF185 function

• Role in diseases beyond those currently studied

Addressing these gaps would provide a more complete understanding of RNF185 biology and its therapeutic potential .

What new technologies could advance RNF185 research?

Several emerging technologies could significantly advance RNF185 research:

Proteomics approaches:

• Proximity labeling methods (BioID, APEX) to identify RNF185 interactome

• Ubiquitinome analysis to identify all RNF185 substrates

• Quantitative mass spectrometry to analyze ubiquitin chain types and linkages

Structural biology techniques:

• Cryo-EM to determine RNF185 structure in complex with substrates

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• AlphaFold or similar AI-based structure prediction to model full-length RNF185

Genetic engineering:

• CRISPR-based screens to identify synthetic lethal interactions with RNF185

• Tissue-specific and inducible knockout models to study physiological functions

• Base editing to introduce and study specific mutations

Drug discovery platforms:

• Small molecule screens for RNF185 modulators

• PROTAC approaches to target RNF185 or enhance its activity

• Peptide-based inhibitors of specific RNF185-substrate interactions

These technologies would provide new insights into RNF185 biology and potentially identify therapeutic approaches for diseases involving RNF185 dysregulation .

RNF185 Domain Structure and Functional Mutants

These structural features and experimental mutants have been essential for dissecting the functional domains of RNF185 and their contributions to its various biological activities .

RNF185 Substrate Specificity

This substrate specificity profile demonstrates that RNF185 selectively targets certain proteins and can mediate different types of ubiquitination with distinct functional outcomes .

Physiological and Pathological Processes Involving RNF185

What is the current state of RNF185 research?

Research on RNF185 has made significant progress in recent years, moving from basic characterization to detailed functional studies in various biological contexts. Key advances include:

• Identification of RNF185 as an important ERAD E3 ligase targeting specific substrates like CFTR

• Discovery of its role in innate immunity through regulation of cGAS

• Elucidation of its tumor suppressor function in prostate cancer

• Recognition of its antiviral activity against SARS-CoV-2

These findings have established RNF185 as a multifunctional protein with important roles in protein quality control, immune regulation, cancer biology, and host-pathogen interactions. The diverse functions of RNF185 highlight the versatility of E3 ubiquitin ligases in cellular physiology and pathology .

How might RNF185 research evolve in the coming years?

Future RNF185 research is likely to focus on several promising directions:

• Detailed structural studies to understand substrate recognition and catalytic mechanisms

• Development of specific modulators (inhibitors or activators) of RNF185 activity

• Exploration of additional substrates and biological functions through proteomics

• Investigation of RNF185 dysregulation in additional disease contexts

• Translation of basic findings into therapeutic applications