RNF114 Human

What is RNF114 and how is it structurally characterized?

RNF114 is a member of the RING (really interesting new gene) domain E3 ubiquitin ligase family. It features a distinctive structure characterized by three zinc-finger domains and one ubiquitin interacting motif (UIM) . The protein efficiently binds both K48- and K63-linked polyubiquitin chains both in vitro and in vivo and possesses E3 ubiquitin ligase activity . RNF114 is expressed as a soluble cytosolic protein, making it accessible for various cellular processes and interactions.

The structural elements of RNF114 are crucial for its function, particularly the RING domain which facilitates its E3 ligase activity. Understanding these structural components provides the foundation for exploring how mutations or polymorphisms might affect its functionality in normal and disease states.

What is the tissue expression pattern of RNF114 in humans?

Real-time PCR analysis has demonstrated that RNF114 exhibits a diverse expression pattern across multiple tissue types. It is clearly expressed in disease-relevant cell types including CD4+ T lymphocytes, dendritic cells, and skin . Beyond immune-related tissues, RNF114 is also expressed in testis, pancreas, kidney, and spleen .

This broad expression profile suggests that RNF114's activity is not restricted to the immune system, indicating potential multifunctional roles across different tissue types. For researchers, this means that when designing studies focused on RNF114, consideration should be given to potential systemic effects beyond the primary tissue of interest, particularly in translational research aiming to modulate RNF114 activity.

How is RNF114 expression regulated at the cellular level?

RNF114 expression is dynamically regulated by several factors, particularly those related to immune responses. The protein can be induced by interferons and synthetic double-stranded RNA (dsRNA) . Time-course experiments have demonstrated that when HEK293T cells are transfected with the synthetic dsRNA analogue polyinosinic:polycytidylic acid [poly(I:C)], RNF114 transcript levels increase in a time-dependent manner .

Additionally, RNF114 expression varies during the cell cycle, with reduced expression at the G1 phase but increased levels at the S and G2/M transition . This cell-cycle dependent regulation suggests that RNF114 elevation may drive the G1 to S transition, potentially contributing to its reported mitogenic function.

What is the relationship between RNF114 and the NF-κB signaling pathway?

RNF114 functions as a negative regulator of NF-κB-dependent transcription through multiple mechanisms. Research has shown that RNF114 stabilizes both the A20 protein and IκBα, which are important negative regulatory molecules that control the NF-κB response . Through these interactions, RNF114 helps fine-tune NF-κB activity in cells stimulated with TNFα or anti-CD3/CD28 antibodies.

Interestingly, the effect of RNF114 on NF-κB activity appears to be dose-dependent, suggesting that regulation of its expression level or its post-translational modification may be crucial for its inhibitory function . Overexpression experiments have demonstrated that RNF114 increases the stability of A20 and IκBα, which could be the primary mechanism by which it regulates the NF-κB pathway .

For researchers investigating inflammatory signaling, these findings position RNF114 as a potential target for modulating immune responses without completely suppressing NF-κB's capacity as a transcriptional activator.

How does RNF114 interact with the protein A20, and what are the functional consequences?

RNF114 physically interacts with A20, an interaction that was initially identified through yeast two-hybrid screening and subsequently confirmed through multiple experimental approaches including pull-down experiments, co-immunoprecipitation assays, and analysis of endogenous protein interactions in Jurkat T cells .

The interaction domain is localized within the E3 ligase domain of A20, suggesting that RNF114 may be part of the A20 ubiquitin-editing complex, similar to other proteins like RNF11 or TAXBP1 . This association increases after TNFα stimulation, likely as a consequence of increased A20 levels following such stimuli .

Functionally, RNF114 promotes the ubiquitylation of A20 without causing its degradation. Instead, this modification appears to alter A20's stability and activity . After T-cell receptor (TCR) stimulation, the form of A20 bound to RNF114 undergoes a mobility shift to a higher molecular weight, suggesting that RNF114 can induce A20 modification under specific conditions .

What role does RNF114 play in the RIG-I/MDA5 innate immune signaling pathway?

RNF114 functions as a positive regulator of the RIG-I/MDA5 innate antiviral response pathway . This signaling cascade is activated by the presence of double-stranded RNA (dsRNA) within the cytoplasm and induces the production of type I interferon through the activation of IRF3 and NF-κB transcription factors .

Experimental evidence shows that RNF114 overexpression enhances NF-κB and IRF3 reporter activity and increases type I and type III interferon mRNA levels . These findings indicate that RNF114 regulates a positive feedback loop that enhances dsRNA-induced production of type I interferons, which are key early mediators of epithelial inflammation .

For researchers studying innate immune responses, this suggests that dysregulation of RNF114 could lead to aberrant RIG-I/MDA5 signaling and overproduction of type I interferons, potentially contributing to inflammatory conditions such as psoriasis.

How is RNF114 linked to psoriasis susceptibility?

Genome-wide association studies (GWAS) have identified RNF114 as a psoriasis susceptibility gene . Specifically, in a GWAS involving 1409 psoriasis patients and 1436 controls, single-nucleotide polymorphisms (SNPs) in RNF114, alongside genes involved in IL-23 signaling, A20, and ABIN-1, showed strong association with psoriasis .

The mechanistic link between RNF114 and psoriasis appears to involve its role in regulating immune signaling. As a positive regulator of the RIG-I/MDA5 pathway, dysregulation of RNF114 may lead to overproduction of type I interferons, which are key mediators of epithelial inflammation . This points to a novel pathogenic pathway in which aberrant RIG-I/MDA5 signaling contributes to the inflammatory processes characteristic of psoriasis.

For clinical researchers, these findings suggest that targeting RNF114 or its downstream effectors might represent a novel therapeutic approach for psoriasis and potentially other inflammatory skin conditions.

What is the evidence linking RNF114 to other autoimmune or inflammatory diseases?

While RNF114 has been most strongly associated with psoriasis, its involvement in immune signaling pathways suggests potential roles in other autoimmune and inflammatory conditions. The protein's interaction with A20 is particularly significant in this context, as polymorphisms within the A20 genomic region predispose individuals to various autoimmune diseases including systemic lupus erythematous and Crohn's disease, in addition to psoriasis .

RNF114's role in regulating NF-κB activity and T-cell function further supports its potential involvement in broader autoimmune processes. As a negative regulator of T-cell activation and a modulator of T-cell apoptosis, RNF114 could influence the development and progression of T-cell-mediated autoimmune conditions .

While direct evidence linking RNF114 to autoimmune diseases beyond psoriasis is still emerging, researchers investigating these conditions should consider RNF114 as a potential contributor to disease mechanisms, particularly in conditions characterized by dysregulated T-cell responses or NF-κB activity.

What are the recommended techniques for studying RNF114 protein interactions?

Several complementary techniques have proven effective for investigating RNF114 protein interactions:

• Yeast Two-Hybrid Screening: This approach was successfully used to identify RNF114 as an A20-interacting protein using a human thymocytes (CD4+ CD8+) cDNA library and a full-length form of A20 .

• GST Pull-down Assays: These can confirm interactions using GST-tagged proteins (e.g., GST-A20) and lysates from cells overexpressing the protein of interest (e.g., FLAG-RNF114) .

• Co-immunoprecipitation (Co-IP): Both with overexpressed and endogenous proteins. For RNF114-A20 interactions, Co-IP experiments in Jurkat T cells using anti-A20 or anti-RNF114-specific antibodies confirmed their association at endogenous levels .

• Stimulation-dependent Interaction Analysis: To examine how protein interactions change upon stimulation, cells can be treated with relevant stimuli (e.g., TNFα or CD3/CD28 antibodies for RNF114-A20 interactions) before Co-IP experiments .

When designing such experiments, researchers should consider that some protein interactions may involve only a fraction of the total protein pool, and antibody efficiency may affect detection sensitivity. Additionally, post-translational modifications may alter interaction dynamics, as observed with A20 after TCR stimulation .

What are effective approaches for studying RNF114 ubiquitination activity?

To investigate RNF114's ubiquitination activity, the following methodologies have been successfully employed:

• In vitro Ubiquitination Assays: These can assess RNF114's E3 ligase activity directly, examining its ability to facilitate ubiquitin transfer to substrate proteins.

• Ubiquitination Analysis in Cell Culture: Cells can be transfected with HA-ubiquitin, myc-A20, and increasing amounts of FLAG-RNF114, followed by immunoprecipitation and Western blot analysis to detect ubiquitinated proteins .

• Stimulus-dependent Ubiquitination: To examine how stimulation affects RNF114-mediated ubiquitination, cells can be treated with relevant stimuli such as PMA/ionomycin before analysis .

• Knockdown Experiments: RNA interference using lentiviral shRNA constructs targeting RNF114 can help determine how reduction of RNF114 affects the ubiquitination and stability of potential substrates like A20 and IκBα .

When interpreting results from these experiments, researchers should consider that RNF114 itself is subject to post-translational modifications, including ubiquitination and SUMOylation, which may affect its activity .

What cell models are most appropriate for studying RNF114 function?

Based on the available research, several cell models have proven valuable for studying RNF114 function:

• Jurkat T Cells: These human T lymphocyte cells have been effectively used to study RNF114's role in T-cell activation, apoptosis, and its interaction with A20 at endogenous levels . They are particularly suitable for investigating RNF114's function in the context of the immune system.

• HEK293/HEK293T Cells: These human embryonic kidney cells have been used for overexpression studies, reporter assays, and to examine RNF114's response to dsRNA stimulation . Their high transfection efficiency makes them useful for biochemical analyses of RNF114 interactions and activity.

• Primary Immune Cells: Since RNF114 is expressed in CD4+ T lymphocytes and dendritic cells, primary cultures of these cell types may provide physiologically relevant insights, though they weren't specifically described in the provided research results.

For functional studies, researchers have successfully employed various stimuli including:

• TNFα (15 ng/ml) for apoptosis studies

• CD3/CD28 antibodies for T-cell activation

• PMA (20 ng/ml)/ionomycin (1 mM) as a "TCR-like" stimulus

• Poly(I:C) to mimic cytosolic dsRNA exposure

When selecting a cell model, researchers should consider the specific aspect of RNF114 function they aim to study, as its roles may vary between different cell types and under different stimulation conditions.

How do post-translational modifications of RNF114 affect its function and interactions?

RNF114 undergoes various post-translational modifications that likely influence its activity and interactions with other proteins. Research has shown that RNF114 is ubiquitinated and potentially SUMOylated . These modifications may alter RNF114's stability, localization, or ability to interact with binding partners and substrates.

The functional significance of these modifications remains an area requiring further investigation. Dose-dependent effects of RNF114 on NF-κB activity suggest that regulation of its expression level or post-translational modification status may be critical determinants of its function . Additionally, the observation that RNF114 can be stabilized or destabilized depending on its expression levels points to complex regulatory mechanisms that warrant detailed examination .

For advanced researchers, investigating the specific E3 ligases or deubiquitinating enzymes that regulate RNF114 modification, as well as mapping the specific sites of modification, could provide valuable insights into how its activity is controlled in different cellular contexts or disease states.

What are the discrepancies in reported effects of RNF114 on NF-κB signaling, and how might they be reconciled?

The literature contains apparent contradictions regarding RNF114's effect on NF-κB activity. While some studies (including those cited in the search results) indicate that RNF114 acts as a negative regulator of NF-κB-dependent transcription by stabilizing inhibitors like A20 and IκBα , other studies have suggested that RNF114 overexpression could have an activating effect on NF-κB activity .

These discrepancies might be reconciled by considering several factors:

• Dose-dependent effects: There is evidence that RNF114's impact is dose-dependent, suggesting that different expression levels may lead to opposing effects .

• Experimental differences: Variations in cell lines, stimuli, luciferase reporters, and RNF114 expression levels across studies could contribute to differing outcomes .

• Context-dependent function: RNF114 activity and function might be tissue and stimulus-dependent, similar to what has been observed with A20 .

• Dual regulatory mechanisms: RNF114 might participate in both positive and negative feedback loops within NF-κB signaling, with the dominant effect determined by specific cellular conditions.

For researchers investigating these discrepancies, carefully controlled experiments comparing RNF114's effects across different expression levels, cell types, and stimulation conditions would be valuable for developing a unified model of its role in NF-κB regulation.

How does RNF114 contribute to the balance between T-cell activation and apoptosis?

The relationship between RNF114 and T-cell function represents a complex area of investigation. Research has demonstrated that RNF114 plays a role in regulating both T-cell activation and apoptosis, but not cell cycle progression in T cells .

Knockdown experiments revealed that when RNF114 expression is reduced in Jurkat T cells:

• There is decreased TNFα-induced apoptosis, as evidenced by reduced Annexin V/7AAD staining and diminished cleavage of PARP and caspases 7 and 9 .

• T-cell activation markers CD69 and CD25 show significant increases after CD3/CD28 or PMA/ionomycin stimulation .

These findings suggest that RNF114 functions as a negative regulator of T-cell activation while promoting apoptotic pathways. This dual role may involve regulation of common signaling nodes, such as NF-κB, which influences both activation and survival pathways in T cells.

For immunology researchers, understanding how RNF114 balances these seemingly opposing functions is crucial, particularly given its association with autoimmune conditions like psoriasis. Future studies might investigate whether this balance is altered in disease states, potentially contributing to hyperactive or persistent T-cell responses characteristic of autoimmune conditions.

What makes RNF114 a potential therapeutic target, and what approaches might be considered?

RNF114 represents an attractive therapeutic target for several reasons. As a modulator of NF-κB activity and T-cell function, it offers the potential to fine-tune immune responses without completely suppressing NF-κB's transcriptional activity . This is particularly valuable since complete NF-κB inhibition can lead to severe immunosuppression and other adverse effects.

Several therapeutic approaches might be considered:

• Small molecule modulators: Compounds that modify RNF114's E3 ligase activity or its interaction with key partners like A20 could provide selective modulation of downstream signaling pathways.

• Targeting specific protein-protein interactions: Disrupting or enhancing specific interactions (e.g., RNF114-A20) might allow for pathway-specific effects rather than global inhibition.

• Gene therapy approaches: In conditions associated with RNF114 dysregulation, correcting expression levels might restore normal immune function.

• Targeting RNF114 post-translational modifications: Modulating the enzymes responsible for RNF114 ubiquitination or other modifications could provide an indirect mechanism to regulate its activity.

Given RNF114's role in the RIG-I/MDA5 pathway and type I interferon production, these approaches might be particularly relevant for treating conditions characterized by excessive interferon signaling, such as psoriasis and potentially other autoimmune diseases .

What are the challenges in translating RNF114 research into clinical applications?

Despite its therapeutic potential, several challenges must be addressed before RNF114-targeted therapies can advance to clinical applications:

• Multifunctional nature: RNF114's involvement in multiple cellular processes and signaling pathways means that targeting it could produce unpredictable effects. Its expression across various tissues suggests potential for off-target effects in non-immune cells .

• Context-dependent activity: Evidence suggests that RNF114's function may be tissue and stimulus-dependent , making it difficult to predict the consequences of modulation across different physiological contexts.

• Dose-dependent effects: The observation that RNF114's impact on NF-κB signaling may be dose-dependent complicates therapeutic approaches, as different levels of inhibition or activation might yield opposing outcomes.

• Incomplete understanding of mechanisms: While progress has been made in understanding RNF114's interactions and functions, significant knowledge gaps remain regarding its complete interactome, substrate specificity, and regulation.

• Genetic variation: As a susceptibility gene for conditions like psoriasis, individual genetic variations in RNF114 might affect response to targeted therapies, necessitating personalized approaches.

Addressing these challenges will require comprehensive studies of RNF114 biology across different cell types and disease models, as well as the development of highly selective modulators that can achieve the desired balance between beneficial and adverse effects.