UBE2F Human

What is UBE2F and what is its role in protein neddylation?

UBE2F is one of two NEDD8-specific E2 conjugating enzymes in mammalian cells (alongside UBE2M/UBC12) that catalyzes the transthiolation reaction in the protein neddylation pathway. The full-length human UBE2F protein consists of 185 amino acids and functions as a unique E2 ubiquitin-conjugating enzyme specifically for neddylation .

Methodologically, investigating UBE2F's function requires structural analysis to understand its key features, which include:

• An N-terminal extension

• A conserved catalytic core domain with distinct features compared to UBE2M

• An offset orientation in its N-terminal α1 helix

• A catalytic cysteine (Cys116) positioned in a loop

• A C-terminal extension forming an α helix rather than a two-stranded β sheet

The interaction between UBE2F and NAE (NEDD8-Activating Enzyme) involves both its N-terminal extension and core domain, with the Met-Leu2-X-Leu4 sequence in UBE2F's N-terminal extension binding to the UBA3 docking groove .

How does UBE2F differ from UBE2M in substrate specificity and structure?

Both UBE2F and UBE2M function as neddylation E2 enzymes but exhibit distinct specificity and structural properties:

These structural and functional differences explain why UBE2F depletion selectively affects CUL5 neddylation without impacting other cullins . This specificity makes UBE2F potentially more selective as a therapeutic target compared to UBE2M.

What evidence demonstrates UBE2F's role in lung cancer progression?

UBE2F plays a significant role in promoting lung cancer progression through multiple mechanisms:

• Clinical correlation: UBE2F is overexpressed in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues and predicts poor patient survival .

• In vitro effects: Ectopic expression of wild-type UBE2F promotes cell survival in lung cancer cells, while the catalytically inactive UBE2F mutant (C116A) suppresses growth and survival, acting in a dominant-negative manner .

• In vivo confirmation: Xenograft models demonstrate that UBE2F-WT promotes tumor growth while UBE2F-C116A mutant suppresses it (p<0.05) .

• Cancer-specific dependency: UBE2F depletion causes suppression of cell survival and induces apoptosis in lung cancer cell lines (H358 and A427) but not in immortalized bronchial epithelial Beas-2B cells, demonstrating tumor cell-specific effects .

• Molecular mechanism: UBE2F promotes cancer cell survival by activating CRL5 to degrade the pro-apoptotic protein NOXA, thereby inhibiting apoptosis .

These findings were established using multiple complementary methodological approaches, including cell viability assays, clonogenic survival assays, xenograft tumor models, and molecular analyses (immunoblotting, immunohistochemistry).

How does the UBE2F/SAG/CRL5 axis regulate NOXA degradation and apoptosis?

The UBE2F/SAG/CRL5 axis regulates NOXA through a unique ubiquitylation and degradation pathway:

• Complex formation: UBE2F forms a tri-complex with SAG and CUL5, as demonstrated by pulldown assays .

• CUL5 neddylation: UBE2F specifically neddylates CUL5, activating the CRL5 E3 ligase complex .

• Novel K11 ubiquitin linkage: Uniquely, activated CRL5 ubiquitylates NOXA via K11 linkage (rather than the conventional K48 linkage) for targeted proteasomal degradation .

• Causal relationship: NOXA knockdown rescues growth inhibition effects caused by UBE2F silencing, indicating that NOXA accumulation drives UBE2F depletion-induced apoptosis .

• Clinical correlation: In lung cancer tissues, high levels of UBE2F and CUL5 correlate with low levels of NOXA and poor patient survival .

This pathway can be experimentally validated through:

• In vivo and in vitro ubiquitylation assays

• Pulldown assays for complex formation

• Expression of K11R ubiquitin mutant (which causes NOXA accumulation)

• siRNA-mediated knockdown for rescue experiments

• Correlation analyses in patient samples

What methodologies are most effective for studying UBE2F activity?

Based on published research, effective methodologies for studying UBE2F activity include:

When studying UBE2F activity, it's essential to include appropriate controls such as:

• Enzymatically dead UBE2F-C116A mutant

• Comparison between cancer and normal cell lines

• Vector-only controls for overexpression studies

• Non-targeting siRNA controls for knockdown experiments

What cellular models are appropriate for UBE2F functional studies?

Selecting appropriate cellular models is crucial for meaningful UBE2F functional studies:

• Lung cancer cell lines:

  • H358 and A427 cells show sensitivity to UBE2F manipulation

  • These models demonstrate clear UBE2F-dependent growth and survival effects

• Normal control cells:

  • Immortalized bronchial epithelial Beas-2B cells serve as appropriate "normal" controls

  • UBE2F depletion shows no significant effect on these cells, highlighting cancer-specific dependency

• Xenograft tumor models:

  • In vivo models using immunodeficient mice implanted with lung cancer cells

  • Effective for validating in vitro findings and testing therapeutic approaches

• Other cancer models:

  • HeLa cells have shown sensitivity to UBE2F knockdown

  • NIH3T3 cells (immortalized mouse fibroblasts) are resistant to UBE2F depletion

When selecting models, researchers should consider:

• Baseline expression levels of UBE2F, CUL5, and SAG

• Dependency on UBE2F for survival (cancer vs. normal)

• Amenability to genetic manipulation (transfection efficiency)

• Relevance to specific cancer types being studied

How does UBE2F specifically recognize and neddylate CUL5 over other cullins?

The molecular basis for UBE2F's specificity toward CUL5 involves several coordinated mechanisms:

• E2-E3 pairing specificity: UBE2F pairs specifically with RBX2/SAG to mediate neddylation of CUL5, while UBE2M couples with RBX1 to neddylate CUL1-4 . This E2-E3 specificity is a primary determinant of cullin substrate selection.

• Structural determinants: UBE2F's unique structural features compared to UBE2M contribute to this specificity:

  • Offset orientation of the N-terminal α1 helix

  • Different positioning of the catalytic cysteine (Cys116)

  • C-terminal α helix instead of a two-stranded β sheet

• DCNL co-E3 preference: UBE2F binds preferentially to the PONY domains of DCNL2 and DCNL3, while UBE2M interacts with DCNL1 . This provides an additional layer of specificity regulation.

To fully elucidate this specificity experimentally, researchers should employ:

• Structural studies of UBE2F-RBX2-CUL5 complexes

• Domain swapping between UBE2F and UBE2M

• Site-directed mutagenesis of interface residues

• In vitro neddylation assays with recombinant proteins

What is the significance of K11 ubiquitin linkage in NOXA degradation?

The discovery that UBE2F/SAG/CRL5 ubiquitylates NOXA via K11 linkage rather than the conventional K48 linkage represents a novel regulatory mechanism with several important implications:

• Alternative degradation signal: While K48-linked ubiquitin chains are the classical signal for proteasomal degradation, this finding demonstrates that K11 linkages can also target proteins for degradation in this context .

• Pathway specificity: The K11 linkage may provide a specific regulatory mechanism for NOXA that distinguishes it from other proteasomal substrates and allows for selective modulation.

• Functional validation: Expression of K11R ubiquitin mutant causes NOXA accumulation and induces apoptosis, which can be rescued by NOXA knockdown . This confirms the functional significance of the K11 linkage.

• Therapeutic implications: Understanding this unique ubiquitylation mechanism may enable more specific therapeutic targeting of the UBE2F/CRL5/NOXA axis without broadly affecting K48-dependent protein degradation.

To study K11 linkage experimentally:

• Use ubiquitin mutants (particularly K11R)

• Employ linkage-specific antibodies

• Perform mass spectrometry analysis of ubiquitin chains

• Design functional rescue experiments with wild-type and K11R ubiquitin

What approaches can be used to target UBE2F for cancer therapy?

Several strategic approaches can be utilized to target UBE2F for cancer therapy:

• Direct targeting of UBE2F:

  • siRNA/shRNA-mediated gene silencing has demonstrated efficacy in preclinical models

  • Development of small molecule inhibitors targeting UBE2F's catalytic site

  • Disruption of UBE2F-specific protein-protein interactions

• Targeting the neddylation pathway:

  • MLN4924 (pevonedistat, NAE inhibitor) blocks the entire neddylation process

  • This approach has shown efficacy in xenograft tumor models with minimal effects on normal tissues

• Exploiting UBE2F selectivity:

  • UBE2F inhibition may offer advantages over broader approaches targeting UBE2M

  • UBE2F depletion selectively suppresses growth of cancer cells without affecting normal cells

• Targeting downstream effectors:

  • Inhibiting the degradation of NOXA or other CRL5 substrates

  • Direct stabilization of pro-apoptotic factors in UBE2F-overexpressing cancers

The search results indicate that "targeting UBE2F, rather than UBE2M, could be a preferred approach for anticancer therapy" due to its cancer-specific effects and narrower substrate specificity .

What biomarkers could predict sensitivity to UBE2F-targeted therapies?

Based on the molecular mechanisms of UBE2F function, several potential biomarkers could predict sensitivity to UBE2F-targeted therapies:

The clinical correlation data shows that in lung cancer tissues, high levels of UBE2F and CUL5 correlate with low levels of NOXA and poor patient survival . This suggests that a panel combining these markers might effectively identify patients most likely to benefit from UBE2F-targeted therapies.

How can researchers distinguish between UBE2F-dependent and UBE2M-dependent neddylation?

Distinguishing between UBE2F-dependent and UBE2M-dependent neddylation is crucial for understanding specific pathway contributions:

• Substrate specificity analysis:

  • UBE2F specifically neddylates CUL5

  • UBE2M neddylates CUL1-4

  • Monitor neddylation status of different cullins by Western blotting

• Selective genetic manipulation:

  • Use siRNA specifically targeting either UBE2F or UBE2M

  • Express dominant-negative mutants (e.g., UBE2F-C116A)

  • Observe cullin-specific effects

• Downstream pathway analysis:

  • UBE2F/CRL5 regulates NOXA via K11 ubiquitin linkage

  • UBE2M/CRL1-4 regulates different substrates (p21, p27, Bim) via K48 linkage

• E3 partner analysis:

  • UBE2F couples with RBX2/SAG

  • UBE2M pairs with RBX1

Research has demonstrated that UBE2F depletion in lung cancer cells specifically eliminates CUL5 neddylation without affecting other cullins, confirming the specificity of this approach .

What are the challenges in developing specific inhibitors of UBE2F?

Developing specific inhibitors of UBE2F presents several technical challenges:

• Structural similarity to other E2 enzymes:

  • UBE2F shares core structural elements with UBE2M and other E2s

  • Achieving selectivity over related enzymes requires detailed structural insights

• Active site conservation:

  • The catalytic cysteine (Cys116) region may be conserved among E2 enzymes

  • Identifying unique binding pockets is essential for selective inhibition

• Protein-protein interaction complexity:

  • UBE2F functions through interactions with multiple partners (NAE, RBX2/SAG, CUL5)

  • Each interaction surface presents potential targeting opportunities but also complications

• Model system requirements:

  • Need for models that accurately recapitulate UBE2F dependency

  • Cancer-specific effects require comparison with normal cell controls

• Validation challenges:

  • Confirming target engagement in cellular contexts

  • Distinguishing UBE2F inhibition from broader neddylation pathway effects

The development of the NAE inhibitor MLN4924, which blocks the entire neddylation pathway upstream of UBE2F, provides proof-of-concept that targeting this pathway has therapeutic potential .

What other potential substrates of UBE2F/CRL5 might exist beyond NOXA?

While NOXA is a well-validated substrate of the UBE2F/CRL5 axis in lung cancer, the broader substrate landscape remains to be fully characterized:

• Known neddylation targets: Research has identified numerous proteins regulated by neddylation pathways , which could provide clues to additional UBE2F/CRL5 substrates:

  • Several proteins regulated via UBE2M-dependent pathways might have UBE2F-dependent counterparts

  • The specific K11 ubiquitin linkage could serve as a signature for identifying CRL5 substrates

• Methodological approaches to identify new substrates:

  • Proteomics analysis of proteins stabilized by UBE2F depletion

  • K11-linkage specific ubiquitin proteomics

  • CRL5 interactome analysis using proximity labeling techniques

  • Comparative studies between UBE2F and UBE2M substrate profiles

• Predicted candidates based on pathway knowledge:

  • Other pro-apoptotic BH3-only proteins

  • Proteins known to be regulated by CUL5-based E3 ligases

  • Factors involved in cancer-specific survival pathways

Identifying the complete repertoire of UBE2F/CRL5 substrates will be crucial for understanding the full spectrum of UBE2F functions and potential therapeutic applications.

How might UBE2F function across different cancer types?

While current evidence primarily focuses on UBE2F's role in lung cancer, several considerations suggest potential relevance across multiple cancer types:

• Lung cancer evidence:

  • UBE2F is overexpressed in NSCLC tissues

  • High UBE2F expression correlates with poor patient survival

  • UBE2F promotes lung cancer growth both in vitro and in vivo

• Other cancer indications:

  • UBE2F knockdown inhibits HeLa cell growth (cervical cancer)

  • UBE2F depletion has no effect on immortalized NIH3T3 cells, suggesting cancer-specific dependency

• Mechanism-based predictions:

  • Cancers with elevated CRL5 activity might depend on UBE2F

  • Tumors with suppressed NOXA or apoptotic resistance could involve UBE2F upregulation

  • Cancers with CUL5 amplification or overexpression might show UBE2F dependency

To investigate UBE2F function across cancer types, researchers should:

• Analyze UBE2F expression across cancer genomics databases

• Perform comparative functional studies in diverse cancer cell lines

• Correlate UBE2F expression with clinical outcomes in multiple cancer types

• Investigate cancer-specific regulatory mechanisms of the UBE2F/CRL5 axis