Recombinant Human RING finger protein 183 (RNF183)

What is RNF183 and what distinguishes it from other RING finger proteins?

RNF183 is a ubiquitin ligase that contains both RING-finger and transmembrane domains. It belongs to the RNF183 family, which includes RNF182, RNF186, and RNF152. These proteins share a similar structure with a RING finger domain (C3HC4) at their N-terminus and transmembrane domains at their C-terminus with high homology . Unlike many RING finger proteins that are predominantly cytosolic, RNF183 localizes to the endoplasmic reticulum (ER) membrane and exhibits classic E3 ligase activities . This localization is particularly important for its role in ER stress response pathways and subsequent cellular signaling cascades. The unique feature of RNF183 is its specific induction during prolonged ER stress, suggesting a specialized role in stress-induced cellular responses rather than housekeeping functions observed in some other RING finger proteins.

How is RNF183 expression regulated at the cellular level?

RNF183 expression is primarily regulated at the post-transcriptional level through mechanisms involving the unfolded protein response (UPR) sensor IRE1α. During sustained ER stress induced by various treatments, RNF183 protein levels increase posttranscriptionally in an IRE1α-dependent manner . Specifically, activated IRE1 reduces the level of microRNA-7 (miR-7), which normally targets RNF183 mRNA for degradation. When miR-7 levels decrease, the stability of RNF183 transcripts increases, leading to higher RNF183 protein expression . This regulatory mechanism explains why RNF183 levels rise specifically under conditions of prolonged ER stress, providing a temporal control mechanism that only activates RNF183-mediated pathways when adaptive UPR mechanisms fail to resolve cellular stress.

What is the subcellular localization of RNF183 and how does this impact its function?

RNF183 is a membrane-spanning protein that primarily localizes to the endoplasmic reticulum (ER) . This localization is critical for its function in ER stress-related pathways. As an ER-resident E3 ubiquitin ligase, RNF183 can directly sense and respond to ER stress conditions. The transmembrane domain anchors RNF183 to the ER membrane, while its RING finger domain extends into the cytosol to interact with target proteins for ubiquitination. This strategic positioning at the ER membrane allows RNF183 to bridge ER stress signaling with downstream effects on mitochondrial apoptosis pathways, particularly through its interaction with and ubiquitination of Bcl-xL . The close proximity of the ER and mitochondria creates a specialized microenvironment that facilitates this signaling interplay, suggesting that RNF183's subcellular localization is essential for its role in determining cell fate during prolonged ER stress.

What are the known substrates of RNF183 and how does it interact with them?

RNF183 has several identified substrates that play critical roles in cell survival and apoptosis pathways:

• Bcl-xL: RNF183 directly interacts with this antiapoptotic member of the Bcl-2 family and polyubiquitinates it for degradation. This interaction promotes apoptosis under conditions of prolonged ER stress by removing the protective effect of Bcl-xL .

• Death Receptor 5 (DR5): RNF183 mediates K63-linked ubiquitination of DR5, a member of the tumor necrosis factor (TNF)-receptor superfamily. This ubiquitination leads to lysosomal degradation of DR5. Paradoxically, while RNF183 promotes DR5 transport to lysosomes, it also enhances TRAIL-induced caspase activation and apoptosis .

• NF-κB pathway components: In colorectal cancer cells, RNF183 activates the NF-κB signaling pathway through P65, stimulating the transcription of the multifunctional chemokine IL-8. This interaction is dependent on RNF183's E3 ubiquitin ligase activity and promotes cell migration and metastasis .

These substrate interactions demonstrate that RNF183 can influence both pro-survival and pro-apoptotic pathways, with the ultimate cellular outcome likely dependent on cellular context and the strength and duration of activating signals.

What types of ubiquitin linkages does RNF183 catalyze and what are their functional consequences?

RNF183 has been demonstrated to catalyze different types of ubiquitin linkages depending on the substrate:

• K48-linked polyubiquitination: RNF183 mediates this type of ubiquitination on Bcl-xL, which typically targets proteins for proteasomal degradation. This leads to decreased levels of this antiapoptotic protein, promoting apoptosis during prolonged ER stress .

• K63-linked ubiquitination: RNF183 catalyzes this non-degradative form of ubiquitination on Death Receptor 5 (DR5), which directs DR5 to lysosomal degradation rather than proteasomal degradation . K63-linked ubiquitination often alters protein localization or function rather than directly signaling for degradation.

The ability of RNF183 to facilitate different ubiquitin linkages provides versatility in its cellular functions, allowing it to impact protein stability, trafficking, and signaling pathway activation. This versatility explains how RNF183 can have context-specific effects that range from promoting apoptosis in ER stress to enhancing metastasis in cancer cells.

How does RNF183 contribute to ER stress-induced apoptosis at the molecular level?

RNF183 plays a crucial role in connecting prolonged ER stress to apoptotic cell death through several molecular mechanisms:

• IRE1α-miR-7-RNF183 axis: During sustained ER stress, activated IRE1α reduces miR-7 levels, stabilizing RNF183 mRNA and increasing RNF183 protein expression .

• Bcl-xL ubiquitination and degradation: Once upregulated, RNF183 interacts with and polyubiquitinates Bcl-xL, targeting this antiapoptotic protein for degradation. The reduction in Bcl-xL levels shifts the balance towards pro-apoptotic factors .

• Mitochondrial pathway activation: The decrease in Bcl-xL likely leads to mitochondrial outer membrane permeabilization, cytochrome c release, and subsequent caspase activation.

This molecular cascade explains how RNF183 functions as a critical decision-making factor in the cell's response to ER stress. When ER stress is prolonged and cannot be resolved through adaptive UPR mechanisms, RNF183 becomes upregulated and pushes the cell toward apoptosis by dismantling antiapoptotic safeguards. Experimental evidence supports this model, as overexpression of RNF183 leads to increased apoptosis while its depletion alleviates ER stress-induced apoptosis .

How is RNF183 involved in inflammatory bowel disease (IBD) pathogenesis?

RNF183 has shown significant implications in inflammatory bowel disease (IBD) pathogenesis through several mechanisms:

• Elevated expression in IBD: Expression levels of RNF183 are increased in patients with IBD, including both Crohn's disease and ulcerative colitis, and in experimental colitis mouse models .

• Early response marker: In dextran sulfate sodium (DSS)-treated IBD mouse models, RNF183 was induced to a greater degree at a very early stage than inflammatory cytokines, suggesting it may be an early driver rather than a consequence of inflammation .

• Cell-type specificity: Fluorescence-activated cell sorting and PCR analysis revealed that RNF183 is specifically expressed in epithelial cells of DSS-treated mice, indicating that increased levels of RNF183 do not result from immune cell infiltration but rather from changes in the intestinal epithelium itself .

• DR5-mediated apoptosis: RNF183 mediates K63-linked ubiquitination and lysosomal degradation of Death Receptor 5 (DR5), but also promotes TRAIL-induced caspase activation and apoptosis . This dual role might contribute to epithelial barrier disruption in IBD, as excessive epithelial cell apoptosis is a hallmark of IBD pathogenesis.

These findings suggest that RNF183 may serve as an early biomarker for IBD and potentially a therapeutic target, as it appears to be involved in the initial phases of disease development before widespread inflammation occurs.

What is the significance of RNF183 in colorectal cancer progression and metastasis?

RNF183 plays a significant role in colorectal cancer (CRC) progression and metastasis, as evidenced by multiple clinical and experimental findings:

These findings establish RNF183 as both a potential prognostic biomarker and a promising therapeutic target in CRC, particularly for preventing metastasis, which is the main cause of CRC-related mortality.

How does RNF183 expression correlate with clinical outcomes in endometrial cancer?

RNF183 demonstrates a complex relationship with endometrial cancer outcomes, showing distinctive expression patterns across different subtypes:

This differential expression pattern and positive prognostic association in endometrial cancer (compared to its negative association in colorectal cancer) highlights the context-dependent nature of RNF183 function. These findings suggest that RNF183 may serve as both a prognostic biomarker and an early diagnostic indicator for endometrial cancer patients, particularly in identifying specific molecular subtypes of the disease.

What are the most effective methods for studying RNF183 E3 ligase activity in vitro?

To effectively study RNF183 E3 ligase activity in vitro, researchers should consider the following methodological approaches:

• Purification of recombinant RNF183 protein:

  • Express RNF183 with appropriate tags (His, GST, etc.) in bacterial or insect cell systems

  • For membrane proteins like RNF183, consider using detergent solubilization or truncated constructs containing just the RING domain for soluble expression

  • Purify using affinity chromatography followed by size exclusion chromatography

• In vitro ubiquitination assays:

  • Components needed: Purified E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), recombinant RNF183, ubiquitin, ATP, and substrate protein (e.g., Bcl-xL or DR5)

  • Detect ubiquitination by Western blotting with anti-ubiquitin antibodies

  • Use ubiquitin mutants (K48R, K63R, etc.) to determine the type of ubiquitin linkage catalyzed

• Substrate identification:

  • Immunoprecipitation coupled with mass spectrometry to identify novel substrates

  • Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling to identify proteins in close proximity to RNF183

  • Yeast two-hybrid screening to identify direct interacting partners

• Functional validation:

  • Site-directed mutagenesis of the RING domain (typically mutation of critical cysteine residues) to create ligase-dead controls

  • Compare wild-type and mutant RNF183 in ubiquitination assays

  • Analyze the effects of these mutations on substrate stability and downstream signaling

These approaches allow for comprehensive characterization of RNF183's enzymatic activity, substrate specificity, and the functional consequences of the ubiquitination events it catalyzes.

What cellular models are most appropriate for investigating RNF183 function in ER stress responses?

When investigating RNF183 function in ER stress responses, selecting appropriate cellular models is crucial for physiologically relevant results:

• Cell line selection:

  • HEK293 cells: Commonly used for initial characterization due to ease of transfection

  • HeLa cells: Often used for ER stress studies with well-characterized UPR pathways

  • Cell lines derived from relevant tissues where RNF183 has physiological functions:

    • Intestinal epithelial cells (e.g., Caco-2, HT-29) for IBD-related studies

    • Colorectal cancer cell lines (e.g., HCT116, SW480) for cancer-related research

    • Endometrial cancer cell lines for studies related to gynecological cancers

• ER stress induction methods:

  • Pharmacological inducers:

    • Tunicamycin (inhibits N-linked glycosylation)

    • Thapsigargin (disrupts calcium homeostasis)

    • DTT or β-mercaptoethanol (disrupt disulfide bond formation)

    • Brefeldin A (inhibits ER-to-Golgi transport)

  • Time-course experiments to distinguish between acute and chronic ER stress responses

• Genetic manipulation approaches:

  • RNF183 overexpression systems (transient and stable)

  • CRISPR/Cas9-mediated knockout of RNF183

  • siRNA or shRNA-mediated knockdown

  • Expression of dominant-negative RNF183 (RING domain mutants)

  • Inducible expression systems to control timing of RNF183 expression

• Readout systems:

  • Monitoring UPR markers (BiP/GRP78, CHOP, XBP1 splicing, ATF6 cleavage)

  • Apoptosis assays (Annexin V/PI staining, caspase activation, PARP cleavage)

  • Live-cell imaging with fluorescent reporters for real-time monitoring

  • Integration with -omics approaches (transcriptomics, proteomics) to capture global cellular responses

These models and approaches allow researchers to investigate the specific role of RNF183 in different phases of the ER stress response and to determine how its E3 ligase activity contributes to cell fate decisions under conditions of prolonged ER stress.

What are the best approaches for studying RNF183 in animal models of disease?

For investigating RNF183 in animal models of disease, researchers should consider these comprehensive approaches:

• Genetic models:

  • Knockout mouse models: Complete RNF183 knockout or conditional knockout using Cre-loxP system for tissue-specific deletion

  • Knockin models: Introduction of specific mutations (e.g., E3 ligase-dead mutations) to study enzymatic function

  • Transgenic overexpression models: Tissue-specific or inducible overexpression of RNF183

  • CRISPR/Cas9-generated models: For rapid generation of genetic alterations

• Disease-specific models:

  • IBD models:

    • DSS-induced colitis: Monitoring RNF183 expression at very early stages (as RNF183 is induced earlier than inflammatory cytokines)

    • TNBS-induced colitis

    • IL-10 knockout spontaneous colitis

  • Cancer models:

    • Colorectal cancer: AOM/DSS chemical induction model

    • Xenograft models using RNF183-manipulated cancer cell lines

    • Patient-derived xenografts (PDX)

  • ER stress models:

    • Tunicamycin injection

    • Diet-induced ER stress (high-fat diet, alcohol)

• Analysis methods:

  • Tissue analysis:

    • Immunohistochemistry for RNF183 expression patterns

    • Cell type-specific expression using FACS sorted populations

    • Laser capture microdissection for analyzing specific tissue regions

  • Molecular analysis:

    • qRT-PCR for mRNA expression

    • Western blotting for protein levels and post-translational modifications

    • Co-immunoprecipitation for protein interactions

    • Ubiquitination assays from tissue lysates

  • Functional readouts:

    • Apoptosis markers (TUNEL staining, caspase activation)

    • Inflammatory markers (cytokine profiling, immune cell infiltration)

    • Disease-specific metrics (intestinal permeability, tumor growth, metastasis)

• Translational approaches:

  • Correlation with human tissue samples

  • Testing therapeutic interventions targeting RNF183 or its pathways

  • Biomarker development based on RNF183 expression

These approaches provide a comprehensive framework for investigating RNF183's role in disease pathogenesis and evaluating its potential as a therapeutic target or biomarker.

How might the dual role of RNF183 in both promoting and inhibiting cell death be reconciled mechanistically?

The seemingly contradictory functions of RNF183 in cell death regulation can be reconciled through several mechanistic explanations:

• Context-dependent substrate selection:

  • In ER stress conditions, RNF183 may preferentially target Bcl-xL for degradation, promoting apoptosis

  • In other contexts, RNF183 might prioritize different substrates with opposing effects on cell survival

  • The cellular milieu (stress conditions, cell type, signal intensity) likely influences which substrates are available and in proximity to RNF183

• Different ubiquitin linkage types:

  • RNF183 catalyzes K48-linked polyubiquitination on some substrates (e.g., Bcl-xL), leading to proteasomal degradation

  • It also mediates K63-linked ubiquitination on others (e.g., DR5), which can affect trafficking rather than stability

  • These different ubiquitin modifications can lead to distinct cellular outcomes

• Temporal dynamics of RNF183 action:

  • Initial RNF183 activation may promote survival mechanisms

  • Prolonged or high-level RNF183 activity might trigger cell death pathways

  • This temporal switch could explain why RNF183 is associated with both adaptive and terminal UPR responses

• Integration with parallel signaling pathways:

  • In cancer contexts, RNF183 activates NF-κB signaling and IL-8 production, promoting survival and metastasis

  • In ER stress contexts, this NF-κB activation might be overridden by stronger pro-apoptotic signals

  • The balance between these competing pathways determines the final cellular outcome

• Cell type-specific effects:

  • Different cell types express distinct complements of RNF183 substrates and pathway components

  • This explains why RNF183 has positive prognostic associations in endometrial cancer but negative associations in colorectal cancer

Understanding these complex interactions requires systems biology approaches that consider the entire network of RNF183 interactions rather than isolated pathways, potentially reconciling its seemingly contradictory functions.

What is the relationship between RNF183 and tumor immune microenvironment in different cancer types?

The relationship between RNF183 and the tumor immune microenvironment appears to be complex and potentially cancer type-specific:

• Negative correlation with tumor purity:

  • In endometrial cancer, RNF183 expression is negatively related to tumor purity

  • This suggests that RNF183 may be associated with higher infiltration of non-tumor cells, including immune cells

• Immune cell infiltration correlations:

  • Analysis using the TIMER database indicates potential correlations between RNF183 expression and infiltrating immune cells in various cancer types

  • This includes correlation with:

    • B cells

    • CD4+ T cells

    • CD8+ T cells

    • Neutrophils

    • Dendritic cells

    • Macrophages

• NF-κB-IL-8 axis in immune modulation:

  • RNF183 activates the NF-κB pathway and stimulates IL-8 production in colorectal cancer

  • IL-8 is a powerful chemokine that recruits and activates neutrophils and other immune cells

  • This mechanism could partly explain how RNF183 influences the tumor immune microenvironment

• Cancer-specific immune signatures:

  • The different prognostic associations of RNF183 in different cancer types suggest cancer-specific effects on immune regulation

  • In endometrial cancer, high RNF183 is associated with favorable prognosis , potentially linked to beneficial immune responses

  • In colorectal cancer, high RNF183 predicts poor outcomes , possibly related to immunosuppressive mechanisms

• Potential mechanisms of immune interaction:

  • Regulation of cell surface receptors through ubiquitination

  • Modulation of inflammatory cytokine production

  • Effects on antigen presentation machinery

  • Influence on cancer cell immunogenicity through ER stress pathways

Further research is needed to fully characterize these relationships and determine whether RNF183 could be targeted to enhance anti-tumor immune responses or overcome immunotherapy resistance in specific cancer types.

How might targeting RNF183 therapeutically affect different pathological conditions?

Targeting RNF183 therapeutically presents both opportunities and challenges across different pathological conditions:

• Potential therapeutic strategies:

  • Small molecule inhibitors: Targeting the RING domain to inhibit E3 ligase activity

  • Protein-protein interaction disruptors: Blocking RNF183 interaction with specific substrates

  • Targeted protein degradation: Using PROTACs or molecular glues to selectively degrade RNF183

  • Gene therapy approaches: siRNA, antisense oligonucleotides, or CRISPR-based technologies

• Expected effects in inflammatory bowel disease:

  • RNF183 inhibition could potentially reduce epithelial cell apoptosis, helping maintain intestinal barrier integrity

  • Given that RNF183 is induced early in experimental colitis , targeting it might prevent disease progression if administered early

  • Possible reduction in TRAIL-induced caspase activation could limit tissue damage

  • Potential concerns: Blocking RNF183 might interfere with normal ER stress responses needed for homeostasis

• Implications for colorectal cancer treatment:

  • Inhibiting RNF183 could potentially reduce NF-κB activation and IL-8 production, limiting cancer cell migration and metastasis

  • Based on association data, RNF183 inhibition might particularly benefit patients with larger tumors, deeper invasion, and more advanced stages

  • Translational table: Expected therapeutic impact based on clinical parameters

    Clinical Parameter	Association with RNF183	Expected Impact of RNF183 Inhibition
    Tumor Size	Positive (P=0.012)	Reduced tumor growth
    Invasive Depth	Positive (P=0.004)	Decreased local invasion
    TNM Stage	Positive (P=0.01)	More effective in advanced stages
    Distant Metastasis	Positive (P=0.009)	Reduced metastatic potential

• Considerations for endometrial cancer:

  • High RNF183 expression is associated with favorable prognosis in endometrial cancer

  • This suggests RNF183 inhibition might be detrimental in this context

  • Potential for biomarker-guided treatment decisions: Using RNF183 expression to guide therapy choices rather than as a direct target

• Balancing therapeutic effects across conditions:

  • Tissue-specific delivery systems could help target RNF183 inhibition to specific affected tissues

  • Intermittent dosing strategies might allow therapeutic benefits while minimizing disruption of normal functions

  • Combination therapies targeting RNF183 alongside other pathway components might provide synergistic benefits with reduced side effects

These therapeutic considerations highlight the importance of context-specific approaches when targeting RNF183, with potential benefits in some conditions but possible detrimental effects in others.

What emerging technologies could advance our understanding of RNF183 biology?

Several cutting-edge technologies hold promise for deeper insights into RNF183 biology:

• Spatial transcriptomics and proteomics:

  • These technologies could reveal the tissue microenvironments where RNF183 is expressed at high resolution

  • Particularly valuable for understanding RNF183's role in complex tissues like intestinal epithelium in IBD or heterogeneous tumor samples

  • Could help explain the negative correlation between RNF183 expression and tumor purity in endometrial cancer

• Single-cell analysis techniques:

  • Single-cell RNA-seq to identify specific cell populations expressing RNF183

  • Single-cell proteomics to detect RNF183 protein levels and modifications

  • Integration of these datasets could reveal cell type-specific RNF183 functions

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize RNF183 localization at the ER membrane

  • Live-cell imaging with FRET-based sensors to monitor RNF183 activity in real-time

  • Correlative light and electron microscopy to understand RNF183's role at ER-mitochondria contact sites

• Structural biology techniques:

  • Cryo-EM to determine RNF183 structure, particularly challenging due to its membrane-embedded nature

  • Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

  • AlphaFold2 and other AI-based structure prediction to model RNF183 interactions with substrates

• Ubiquitinome analysis:

  • Global profiling of ubiquitinated proteins in RNF183-manipulated systems

  • Ubiquitin remnant profiling to identify specific lysine residues modified by RNF183

  • Linkage-specific ubiquitin antibodies to distinguish between different ubiquitin chain topologies

• CRISPR-based functional genomics:

  • CRISPR activation/inhibition screens to identify genes that modify RNF183 phenotypes

  • Base editing to introduce specific point mutations in the RING domain or substrate-binding regions

  • CRISPR-Cas13 for temporal control of RNF183 expression

These technologies would provide unprecedented insights into RNF183's molecular mechanisms, potentially revealing new therapeutic opportunities while addressing fundamental questions about this protein's complex biology.

How can contradictory findings about RNF183 in different cancer types be reconciled?

The seemingly contradictory findings regarding RNF183's role across different cancer types require systematic investigation to reconcile:

• Molecular context analysis:

  • Compare the molecular landscapes of cancers where RNF183 has opposing effects:

    • Colorectal cancer: High RNF183 associated with poor prognosis

    • Endometrial cancer: High RNF183 linked to favorable prognosis

  • Investigate differences in:

    • Common mutations (e.g., TP53 status - RNF183 is higher in TP53-Non-Mutant endometrial cancers)

    • Signaling pathway activation states

    • Substrate availability and expression levels

• Comprehensive substrate profiling:

  • Perform cancer type-specific immunoprecipitation-mass spectrometry to identify tissue-specific RNF183 interactors

  • Compare ubiquitinome profiles between different cancer types with RNF183 manipulation

  • This could reveal cancer-specific substrates explaining divergent effects

• Integration with cancer subtype information:

  • In endometrial cancer, RNF183 expression is higher in endometrioid vs. non-endometrioid subtypes

  • Similar subtype-specific analysis in other cancers could reveal patterns explaining contradictory findings

  • Create a subtype-specific expression matrix across cancer types

• Immune microenvironment correlation:

  • Compare RNF183's correlation with immune cell infiltration patterns across cancer types

  • Different immune contexts might explain why the same molecular mechanism has opposite effects on patient outcomes

• Experimental validation approaches:

  • Cross-cancer cell line panels treated with identical RNF183 manipulations

  • Patient-derived organoids from multiple cancer types to preserve tissue-specific contexts

  • Xenograft models with RNF183 overexpression/knockdown in different cancer types

• Multi-omics data integration:

  • Leverage existing TCGA, CPTAC, and other cancer databases

  • Apply machine learning approaches to identify context-specific RNF183 interaction networks

  • Use this information to build predictive models of when RNF183 will have tumor-promoting vs. tumor-suppressing effects

This systematic approach would help establish a unified framework explaining RNF183's context-dependent effects across cancer types, potentially leading to more precise biomarker applications and therapeutic strategies.

What is the evolutionary significance of RNF183 and how is it conserved across species?

Understanding the evolutionary history of RNF183 provides valuable insights into its fundamental biological functions: