UBE2E3 Human

What is UBE2E3 and what is its primary cellular function?

UBE2E3 is a ubiquitin-conjugating enzyme (E2) that works with multiple E3 ligases to facilitate the conjugation of monoubiquitin onto various substrate proteins. This enzyme is highly conserved across species, with mouse and human UBE2E3 protein sequences being identical, underscoring its evolutionary importance . As an E2 enzyme, UBE2E3 functions as an essential component of the ubiquitin-proteasome system (UPS), which regulates protein degradation, cellular signaling, and various other cellular processes.

The primary cellular function of UBE2E3 appears to be maintaining proper cell proliferation and homeostasis. Research has demonstrated that UBE2E3 is essential for cell cycle progression, as its depletion causes robust increases in p27Kip1 levels, leading to cell cycle exit . Additionally, UBE2E3 plays crucial roles in maintaining mitochondrial network integrity and proper functioning of the antistress transcription factor Nrf2, both of which are critical for cellular health and preventing premature senescence .

How is UBE2E3 structurally and functionally related to other ubiquitin-conjugating enzymes?

UBE2E3 belongs to the UBE2E subfamily of E2 enzymes, which also includes UBE2E1 and UBE2E2. While the search results don't provide detailed structural information, we know that UBE2E3 functions as a partner to multiple E3 ligases to conjugate monoubiquitin onto substrates . Unlike some E2 enzymes that form polyubiquitin chains, UBE2E3 specifically facilitates monoubiquitination, which often serves signaling functions rather than targeting proteins for degradation.

The UBE2E family is distinct from other ubiquitin-conjugating enzymes like UBE2B, UBE2G2, and UBE2K, which were identified as survival-related genes in cancer research . While all these enzymes participate in ubiquitination, they likely have different substrate specificities, interaction partners, and cellular functions. Research suggests that certain E2 proteins, including some in the UBE2 family, have highly specific TM-E3-RING interaction partners , indicating specialized roles within the ubiquitin system.

Where is UBE2E3 predominantly localized within human cells, and how does this relate to its function?

UBE2E3 predominantly localizes to the nucleus in human cells, as demonstrated in studies with telomerase-immortalized human retinal pigment epithelial (RPE) cells . This nuclear localization is significant because it suggests that UBE2E3's primary ubiquitination targets are likely nuclear proteins involved in processes such as transcriptional regulation, DNA replication, or cell cycle control.

The nuclear localization of UBE2E3 aligns with its role in cell proliferation, as many cell cycle regulators and transcription factors that control proliferation reside in the nucleus. For instance, UBE2E3 depletion leads to increased nuclear p53 levels (approximately 2.5-fold) and elevated expression of the cell cycle inhibitor p21CIP1/WAF1 , indicating that UBE2E3 may normally suppress these anti-proliferative factors through its nuclear activity. Additionally, UBE2E3's role in maintaining proper Nrf2 activity could be facilitated by its nuclear localization, as Nrf2 functions as a transcription factor within the nucleus.

What are the most effective methods for depleting UBE2E3 in cellular models?

Based on the search results, RNA interference using small interfering RNA (siRNA) has been established as an effective method for depleting UBE2E3 in cellular models. Researchers have successfully used siRNA targeting UBE2E3 in human retinal pigment epithelial (RPE-1) cells, consistently achieving knockdown efficiency of 70-80% at the mRNA level .

The methodology typically involves:

• Design of siRNA oligos specifically targeting UBE2E3 mRNA

• Transfection of cells using appropriate reagents (e.g., Lipofectamine 2000)

• Incubation period (typically 3-6 days) to allow for protein depletion

• Verification of knockdown efficiency by RT-PCR and/or Western blotting

To ensure specificity of the observed phenotypes, rescue experiments are essential. These involve transfecting cells with an siRNA-impervious UBE2E3 cDNA in which the wobble positions of codons within the siRNA target sequence are mutated without altering the amino acid sequence . Successful rescue of the knockdown phenotype confirms that the observed effects are specifically due to UBE2E3 depletion rather than off-target effects.

How can researchers validate UBE2E3 knockdown efficiency and specificity?

Validating UBE2E3 knockdown requires multiple complementary approaches to assess both efficiency and specificity:

• mRNA quantification: Semi-quantitative or quantitative RT-PCR using UBE2E3-specific primers can measure reductions in mRNA levels. The research indicates primers with sequences 5′-GTGATAGGCAAAGGTCCGATGATG-3′ and 5′-AGTGATAGATTCTGGTGCGGAAAG-3′ have been used successfully .

• Protein level assessment: Western blotting with UBE2E3-specific antibodies can confirm reduction at the protein level. Immunofluorescence may also be used to visualize decreased UBE2E3 expression in intact cells.

• Phenotypic assessment: Observing expected phenotypes like cell cycle exit, increased p27Kip1 levels, loss of Ki-67, and increased cell area provides functional validation of knockdown .

• Rescue experiments: The gold standard for specificity validation is the rescue experiment, where an siRNA-resistant UBE2E3 construct is expressed in knockdown cells. The research demonstrates that expression of siRNA-impervious UBE2E3 cDNA can rescue the p27Kip1 accumulation phenotype, confirming knockdown specificity .

• Control siRNAs: Including non-targeting control siRNAs in experiments ensures that observed effects are not due to the transfection process or general RNA interference machinery activation.

What in vivo models are available for studying UBE2E3 function?

The search results indicate that mouse models have been developed for studying the in vivo function of UbcM2, the mouse counterpart of human UBE2E3. Specifically, a mouse strain harboring a disrupted allele of UbcM2 with the coding sequence for β-galactosidase has been created . This reporter model allows tracking of the developmental expression patterns of UbcM2 in various tissues, including murine RPE cells.

This mouse model revealed that UbcM2 expression is transcriptionally downregulated during RPE maturation , suggesting a developmental role for the enzyme in the transition from proliferation to differentiation. The β-galactosidase reporter driven by the endogenous UbcM2 promoter provides a valuable tool for studying tissue-specific and developmental expression patterns.

For researchers interested in studying UBE2E3 function in vivo, these mouse models offer several advantages:

• Ability to track native expression patterns during development

• Potential for tissue-specific analysis of UBE2E3/UbcM2 function

• Opportunity to study the physiological impacts of UBE2E3/UbcM2 deficiency at the organismal level

How does UBE2E3 depletion induce cellular senescence, and what are the key markers of this phenotype?

UBE2E3 depletion induces a distinct form of cellular senescence that is independent of DNA damage. The mechanism appears to involve disruption of multiple cellular processes, including cell cycle regulation, mitochondrial homeostasis, and stress response pathways . Upon UBE2E3 knockdown, cells exhibit a constellation of senescence markers:

• Morphological changes: Cells become enlarged and flattened, with a doubling in cell area .

• Cell cycle markers: Loss of proliferation marker Ki-67, increased p27Kip1 levels , and elevated expression of p16INK4a and p21CIP1/WAF1 .

• Senescence-associated β-galactosidase (SA-β-gal): Increased activity, a canonical marker of senescent cells .

• Nuclear changes: Reduced incorporation of DNA counterstains like DAPI and Hoechst, redistribution of lamin B from the nuclear envelope to punctate nucleoplasmic distribution, and decreased lamin B1 mRNA expression .

• Secretory phenotype: Increased secretion of the pro-inflammatory cytokine IL-6 (5-fold greater than control cells) .

• High-mobility group box 1 (HMGB1): Decreased intracellular content, consistent with senescent cells .

This senescence signature is distinct from DNA damage-induced senescence, as evidenced by minimal phospho-γH2A.X foci formation in UBE2E3-depleted cells compared to etoposide-treated cells .

What is the relationship between UBE2E3, mitochondrial function, and cellular senescence?

UBE2E3 depletion causes significant alterations in mitochondrial function and dynamics that contribute to cellular senescence. The relationship involves several interconnected aspects:

These mitochondrial changes in UBE2E3-depleted cells appear to represent a response to cellular stress rather than direct mitochondrial damage, as UBE2E3 knockdown did not detectably damage mitochondrial integrity based on MitoSOX and JC-1 assays .

How does UBE2E3 depletion-induced senescence differ from other forms of cellular senescence?

UBE2E3 depletion induces a form of cellular senescence with a distinct profile that differentiates it from other established senescence pathways:

UBE2E3 depletion-induced senescence appears to be independent of DNA damage, as evidenced by minimal phospho-γH2A.X foci . It depends on the expression of tumor suppressor p16INK4a and nuclear expression of p53 and p21CIP1/WAF1, and is characterized by increased mitochondrial and lysosomal mass and enhanced basal autophagic flux .

A distinguishing feature is the SASP (Senescence-Associated Secretory Phenotype) profile. While etoposide-treated cells (DDR senescence) showed increases in IL-1β, IL-6, IL-8, CXCL1, and CXCL2, UBE2E3-depleted cells only showed an increase in IL-1β, and this increase was markedly suppressed compared to etoposide-treated cells . This suggests that UBE2E3 depletion induces a senescence with a distinct inflammatory signature.

Why are RPE cells commonly used as a model system for studying UBE2E3 function?

RPE cells represent an excellent model system for studying UBE2E3 function for several compelling reasons:

• Non-transformed cellular model: The telomerase-immortalized human RPE cells (RPE-1) used in UBE2E3 studies are human diploid, non-transformed, contact-inhibited epithelial cells that maintain many characteristics of parental RPE cells, including expression of RPE-specific proteins . This makes them more physiologically relevant than cancer cell lines.

• Consistent knockdown efficiency: RPE-1 cells reproducibly yield knockdown efficiency of UBE2E3 by 70-80% at the mRNA level , making them technically suitable for depletion studies.

• Clear phenotypic responses: UBE2E3 depletion produces robust and easily measurable phenotypes in RPE cells, including cell cycle exit, increased p27Kip1 levels, loss of Ki-67, and increased cell area , facilitating experimental analysis.

• Developmental relevance: Studies in mice demonstrate that UbcM2 (mouse UBE2E3) is transcriptionally downregulated during RPE maturation , suggesting a physiological role for this enzyme in RPE development.

• Intact chromosome count: RPE-1 cells have a modal chromosome count of 46 , maintaining normal human karyotype and avoiding the chromosomal instability common in cancer cell lines.

These characteristics make RPE cells an ideal system for studying fundamental aspects of UBE2E3 biology, particularly its roles in cell proliferation, senescence, and development.

What specific experimental techniques are used to study UBE2E3 in RPE cells?

Research on UBE2E3 in RPE cells employs several specialized techniques:

• UBE2E3 localization: Immunofluorescence using UBE2E3-specific antibodies to determine subcellular localization in RPE cells .

• siRNA-mediated knockdown: Transfection of RPE cells with siRNA targeting UBE2E3, followed by a 3-6 day incubation period to allow for protein depletion .

• Rescue experiments: Transfection with plasmids encoding siRNA-impervious UBE2E3 cDNA to confirm phenotype specificity. These constructs contain mutations in the wobble positions of codons within the siRNA target sequences, generated using site-directed mutagenesis kits like Quickchange (Stratagene) .

• Proliferation markers: Immunolabeling for Ki-67 to assess proliferation status following UBE2E3 manipulation .

• Cell cycle regulators: Immunolabeling for p27Kip1 to evaluate cell cycle exit and assessment of p53, p21CIP1/WAF1, and p16INK4a levels .

• RNA analysis: Extraction of RNA using TRIzol reagent, followed by first-strand synthesis with PowerScript and oligo dT primers, and semi-quantitative PCR with gene-specific primers .

• Morphological assessment: Cell area measurements and microscopic evaluation of cellular morphology to identify senescence-associated changes .

• Senescence markers: SA-β-gal staining, DAPI/Hoechst incorporation, lamin B distribution, and HMGB1 content assessment .

These techniques collectively provide a comprehensive toolkit for investigating UBE2E3 function in RPE cell proliferation, differentiation, and senescence.

How does UBE2E3 expression change during RPE development, and what are the implications?

Studies using a mouse model with a disrupted UbcM2 (mouse UBE2E3) allele containing β-galactosidase reveal that UbcM2 expression is transcriptionally downregulated during RPE maturation in vivo . This finding has several important implications:

• Developmental regulation: The transcriptional downregulation of UbcM2 during RPE development suggests it plays a specific role in the early stages of RPE formation when cells are actively proliferating.

• Proliferation to differentiation transition: The correlation between UbcM2 downregulation and RPE maturation implicates the enzyme in the developmental transition from a state of proliferation to one of differentiation . This aligns with in vitro findings that UBE2E3 depletion causes cell cycle exit in RPE cells.

• Tissue-specific regulation: The use of the heterologous reporter (β-galactosidase) driven by the endogenous UbcM2 promoter allowed researchers to track expression patterns specifically in developing RPE, suggesting tissue-specific regulatory mechanisms.

• Potential role in disease: In conditions where RPE cells inappropriately re-enter the cell cycle (as in some retinal diseases), UBE2E3/UbcM2 expression levels may be altered, suggesting potential diagnostic or therapeutic relevance.

These findings highlight UBE2E3's important role beyond basic cellular processes, positioning it as a developmentally regulated enzyme involved in the maturation of specific cell types like RPE. This developmental perspective provides context for understanding UBE2E3's function in cellular proliferation and senescence prevention.

How does UBE2E3 integrate with the broader ubiquitin-proteasome system and autophagy pathways?

UBE2E3 appears to function at the intersection of the ubiquitin-proteasome system (UPS) and autophagy pathways, highlighting the complex interplay between these two major cellular degradation systems:

• Ubiquitination activity: As an E2 enzyme, UBE2E3 partners with multiple E3 ligases to conjugate monoubiquitin onto substrates . This monoubiquitination can serve diverse signaling functions beyond just targeting proteins for proteasomal degradation.

• Impact on autophagic flux: UBE2E3 depletion alters autophagic flux, as demonstrated using GFP-tagged LC3, a reliable proxy for monitoring autophagy . This suggests that UBE2E3-mediated ubiquitination may regulate autophagy initiation or progression.

• Mitophagy regulation: UBE2E3 knockdown leads to dramatic increases in lysosome-associated mitochondria , indicating enhanced mitophagy. This connects UBE2E3 to selective autophagy pathways targeting mitochondria.

• UPS-autophagy crosstalk: The research notes that ubiquitin can decorate damaged mitochondria with polyubiquitin chains that serve as initiating signals for mitochondrial elimination , highlighting how the ubiquitin system (in which UBE2E3 participates) can trigger selective autophagy.

• System integration: The authors specifically note that their discoveries "highlight the extent to which Ub integrates the UPS and autophagy systems, and it is within this context that we have been investigating the metazoan enzyme, UBE2E3" , positioning UBE2E3 as a key player in the coordination between these systems.

This integration suggests that disruption of UBE2E3 function may contribute to pathological conditions through dysregulation of proteostasis, potentially linking UBE2E3 to diseases associated with protein aggregation or impaired cellular quality control.

What is known about the specific substrates of UBE2E3 and how they relate to its cellular functions?

The search results provide limited information about specific UBE2E3 substrates, but offer some insights into potential targets based on cellular phenotypes:

• Cell cycle regulators: The increase in p27Kip1 levels upon UBE2E3 depletion suggests that this cyclin-dependent kinase inhibitor might be directly or indirectly regulated by UBE2E3-mediated ubiquitination. Similarly, the effects on p53, p21CIP1/WAF1, and p16INK4a levels point to potential regulatory relationships.

• Mitochondrial proteins: Given UBE2E3's role in maintaining mitochondrial network integrity , it likely targets proteins involved in mitochondrial dynamics, morphology, or function. The dramatic redistribution of the mitochondrial network upon UBE2E3 depletion suggests it may ubiquitinate proteins controlling mitochondrial distribution.

• Nrf2 pathway components: UBE2E3 depletion causes a reduction in the activity of the master anti-stress transcription factor Nrf2 . While the exact mechanism is not detailed, this suggests UBE2E3 may ubiquitinate Nrf2 itself or regulators of the Nrf2 pathway.

• Nuclear proteins: Given UBE2E3's nuclear localization , its substrates likely include nuclear proteins involved in transcriptional regulation, DNA replication, or nuclear architecture.

Research to identify specific UBE2E3 substrates would be valuable for elucidating the molecular mechanisms underlying its cellular functions. Approaches might include proteomic analysis comparing ubiquitination patterns in control versus UBE2E3-depleted cells, or identification of proteins that physically interact with UBE2E3 through techniques like co-immunoprecipitation or proximity labeling.

How might UBE2E3 dysfunction contribute to human diseases, and what therapeutic implications does this suggest?

While the search results don't directly address disease associations, they provide insights into potential pathological roles for UBE2E3 dysfunction:

• Cellular senescence and aging: The finding that UBE2E3 depletion induces senescence suggests that dysregulation of this enzyme could contribute to premature cellular aging or senescence-associated diseases. The authors specifically note that "a disruption of the UPS machinery and Ub homeostasis might underlie or contribute to pathological senescence in certain tissues" .

• Retinal diseases: Given UBE2E3's importance in RPE cell proliferation and development , its dysfunction could potentially contribute to retinal pathologies involving RPE cells, such as age-related macular degeneration (AMD) or proliferative vitreoretinopathy.

• Cancer: The relationship between UBE2E3 and cell cycle regulation suggests potential roles in cancer. While UBE2E3 itself wasn't identified among the survival-related ubiquitination genes in cancer , related E2 enzymes (UBE2B, UBE2E2, UBE2G2, UBE2K) were included in a risk model for cancer prognosis .

• Mitochondrial disorders: UBE2E3's role in maintaining mitochondrial homeostasis suggests its dysfunction might contribute to diseases involving mitochondrial dysfunction or abnormal mitophagy.

Therapeutic implications might include:

• Targeting senescence: Modulating UBE2E3 activity could potentially influence cellular senescence, which has implications for senolytic therapies aimed at eliminating senescent cells.

• RPE regeneration: Understanding UBE2E3's role in RPE development could inform strategies for RPE regeneration in retinal diseases.

• Cancer therapies: The differential importance of ubiquitination-related genes, potentially including UBE2E3, in cancer survival suggests they could be therapeutic targets in precision oncology.

• Small molecule modulators: Developing compounds that specifically modulate UBE2E3 activity could provide therapeutic approaches for diseases involving its dysregulation.

Further research is needed to establish direct links between UBE2E3 dysfunction and specific human diseases, which would clarify its potential as a therapeutic target.

What are the common challenges in studying UBE2E3 and how can researchers overcome them?

Based on the search results, several challenges can be inferred in UBE2E3 research:

• Ensuring specific knockdown: RNA interference can have off-target effects. This challenge can be addressed through rescue experiments using siRNA-impervious UBE2E3 constructs , as well as using multiple siRNA sequences targeting different regions of UBE2E3 mRNA to verify consistency of phenotypes.

• Distinguishing direct from indirect effects: Given UBE2E3's role in the ubiquitin system, its depletion could have wide-ranging effects through altered proteostasis. Researchers can address this by:

  • Identifying direct interaction partners and substrates through techniques like BioID or IP-MS

  • Using catalytically inactive UBE2E3 mutants to separate scaffolding from enzymatic functions

  • Employing time-course experiments to determine the sequence of events following UBE2E3 depletion

• Temporal considerations: UBE2E3 depletion phenotypes develop over several days , requiring careful experimental planning. Extended cell culture periods increase the risk of confounding factors like contact inhibition or nutrient depletion. Researchers should include appropriate time-matched controls and consider inducible knockdown systems for temporal control.

• Translating between model systems: While mouse UbcM2 and human UBE2E3 are identical in protein sequence , regulatory mechanisms may differ between species. Researchers should validate key findings across multiple model systems when possible.

• Detecting monoubiquitination: As UBE2E3 primarily facilitates monoubiquitination rather than polyubiquitination , detecting its activity can be challenging. Techniques optimized for detecting single ubiquitin modifications, such as specialized antibodies or mass spectrometry approaches, may be necessary.

How can researchers distinguish between the different functions of UBE2E3 in cell cycle regulation, mitochondrial homeostasis, and senescence?

Distinguishing between UBE2E3's various functions requires carefully designed experiments and analytical approaches:

• Genetic complementation with domain mutants: Researchers can create UBE2E3 constructs with mutations in specific domains or interaction surfaces and test which functions they rescue in UBE2E3-depleted cells. This can help map different functions to distinct structural features of the protein.

• Temporal analysis: Time-course experiments following UBE2E3 depletion can reveal the sequence of phenotypic changes. For example, if mitochondrial alterations consistently precede cell cycle changes, this would suggest the mitochondrial function may be primary.

• Selective rescue experiments: Expression of other genes involved in specific pathways (e.g., mitochondrial dynamics regulators, cell cycle components, or anti-senescence factors) can help determine which downstream effects are causal and which are consequential.

• Pathway inhibitors: Pharmacological inhibitors targeting specific pathways (e.g., cell cycle, mitochondrial function, senescence mediators) can be used in combination with UBE2E3 depletion to dissect interdependencies.

• Single-cell analysis: Techniques like single-cell RNA-seq or imaging-based single-cell analysis can reveal whether all phenotypes occur uniformly across the cell population or if there are distinct subpopulations with different primary responses to UBE2E3 depletion.

• Substrate identification: Identifying specific UBE2E3 substrates in each pathway would provide mechanistic insights into how UBE2E3 separately regulates these different cellular processes.

A systematic approach combining these strategies would help determine whether UBE2E3's roles in cell cycle regulation, mitochondrial homeostasis, and senescence prevention represent independent functions or are interconnected aspects of a unified cellular response.

What new technologies and approaches might advance our understanding of UBE2E3 function in the near future?

Several emerging technologies and approaches could significantly advance UBE2E3 research:

• CRISPR-Cas9 genome editing: Generation of UBE2E3 knockout or knock-in cell lines and animal models would provide cleaner genetic models than RNAi. CRISPR screening approaches could also identify genetic interactions and modifiers of UBE2E3 function.

• Proximity labeling proteomics: Techniques like BioID, TurboID, or APEX2 could identify proteins that physically interact with or are in close proximity to UBE2E3 in living cells, helping to map its functional networks and potential substrates.

• Ubiquitinome analysis: Advanced mass spectrometry approaches for comprehensive mapping of ubiquitination sites could identify UBE2E3-dependent modifications by comparing wild-type and UBE2E3-depleted cells.

• Single-cell multi-omics: Combining single-cell transcriptomics, proteomics, and metabolomics could provide integrated views of how UBE2E3 regulates multiple cellular systems simultaneously.

• Live-cell imaging of ubiquitination: Fluorescent ubiquitin sensors could enable real-time visualization of UBE2E3-mediated ubiquitination events in living cells.

• Structural biology approaches: Cryo-EM or X-ray crystallography of UBE2E3 in complex with E3 partners and substrates would provide mechanistic insights into its function and specificity.

• Tissue-specific conditional knockout models: These would allow investigation of UBE2E3's role in specific tissues during development and in disease models without the confounding effects of embryonic lethality if constitutive knockout is detrimental.

• Patient-derived cells and tissues: Analysis of UBE2E3 expression, localization, and function in patient-derived materials could reveal disease-relevant alterations and provide translational insights.

These approaches, individually or in combination, could address current knowledge gaps and provide a more comprehensive understanding of UBE2E3's roles in cellular homeostasis and disease.