UBE2E1 Human

What is UBE2E1 and what is its primary function in human cells?

UBE2E1 (also known as UBCH6) is a class III ubiquitin-conjugating enzyme (E2) that participates in the ubiquitination cascade. It functions by transferring activated ubiquitin from E1 enzymes to protein substrates, often with the assistance of E3 ligases. UBE2E1 plays crucial roles in protein degradation pathways and cellular signaling .

Human class III E2s, including UBE2E1, have been shown to engage in complexes with damaged DNA binding protein 1 and de-etiolated 1, which are substrate adaptor components for CUL4a E3 ligases . This suggests a role for UBE2E1 in DNA damage response pathways.

What is the tissue-specific expression pattern of UBE2E1?

UBE2E1 demonstrates preferential expression in the cytoplasm of slow-twitch muscle fibers (type I fibers) . This specific expression pattern suggests specialized functions in maintaining the integrity of slow-twitch muscle tissue. Immunohistochemistry studies have shown that UBE2E1 can be detected in muscle cross-sections using a rabbit polyclonal antibody against UBE2E1 (UBCH6) .

When comparing different muscle types, UBE2E1 shows higher expression in muscles predominantly composed of slow-twitch fibers, such as the soleus muscle, compared to fast-twitch fiber-dominant muscles like the extensor digitorum longus (EDL) .

What is known about the structural characteristics of UBE2E1?

The structure of UBE2E1 has been determined through crystallography as evidenced by the RCSB database entry 8IYA, which describes UBE2E1 in complex with a SETDB1-derived peptide . This structural information has revealed important insights about the mechanism of UBE2E1-mediated ubiquitination.

The structural analysis has been particularly valuable in understanding how UBE2E1 can perform E3-independent ubiquitination, which is a unique capability among E2 enzymes. This knowledge has led to the development of an E3-free enzymatic strategy called SUE1 (sequence-dependent ubiquitination using UBE2E1) for generating ubiquitinated proteins with customized specifications .

How does UBE2E1 contribute to muscle homeostasis?

UBE2E1 plays a protective role against exacerbated muscle atrophy, particularly under conditions of dexamethasone (Dex) treatment . Research has demonstrated that UBE2E1, in conjunction with the E3 ligase MuRF1, can target α-actin for degradation, showing a 58% reduction in α-actin levels when co-expressed with MuRF1 .

Interestingly, UBE2E1 shows selectivity in protein targeting. While it facilitates α-actin degradation (a protein present in all muscle fiber types), it does not appear to direct the degradation of MHCIIa (type IIA myosin heavy chain) . This substrate specificity may explain its preferential expression in slow-twitch fibers, which do not contain MHCIIa.

In vivo knockdown studies of UBE2E1 in mice have provided valuable insights into its function in muscle homeostasis, with analysis performed using both one-way and three-way ANOVA to distinguish the specific effects of UBE2E1 knockdown from other variables .

What role does UBE2E1 play in ciliary function and Wnt signaling?

UBE2E1 is involved in the regulation of canonical Wnt signaling through its interaction with MKS1, a ciliary protein. This functional interaction occurs at the ciliary base, where UBE2E1 participates in the processing of phosphorylated β-catenin .

The loss of UBE2E1 recapitulates the ciliary and Wnt signaling phenotypes observed during the loss of MKS1, suggesting a critical role for UBE2E1 in ciliary function . Additionally, levels of UBE2E1 and MKS1 appear to be co-dependent, with UBE2E1 mediating both regulatory and degradative ubiquitination of MKS1 .

This connection between UBE2E1 and ciliary function provides a mechanistic explanation for the long-recognized link between ciliary dysfunction and increased canonical Wnt signaling .

Can UBE2E1 function in the absence of E3 ligases?

One of the most significant recent discoveries is that UBE2E1 can catalyze ubiquitination reactions independently of E3 ligases in specific contexts . The structure of UBE2E1 in complex with a SETDB1-derived peptide has revealed the mechanism underlying this E3-independent ubiquitination capability .

This discovery has led to the development of SUE1 (sequence-dependent ubiquitination using UBE2E1), an enzymatic strategy that efficiently generates ubiquitinated proteins with customized ubiquitination sites, ubiquitin chain linkages, and chain lengths without requiring E3 ligases .

This E3-independent activity represents a unique aspect of UBE2E1 function and has significant implications for both understanding naturally occurring ubiquitination processes and developing research tools for studying ubiquitination.

What experimental approaches are most effective for studying UBE2E1 interactions?

Based on the research literature, several methodological approaches have proven valuable for studying UBE2E1 interactions:

Table 1: Experimental Methods for Studying UBE2E1 Interactions

Researchers should select methods based on their specific research questions, with combined approaches often yielding the most comprehensive insights.

How can UBE2E1 be utilized for generating customized ubiquitinated proteins?

The discovery of UBE2E1's ability to perform sequence-dependent, E3-independent ubiquitination has led to the development of the SUE1 strategy for generating customized ubiquitinated proteins . This methodological approach offers several advantages:

• Site-specific ubiquitination: The method allows researchers to target specific lysine residues for ubiquitination.

• Custom ubiquitin chain linkages: Researchers can specify the type of linkage (K48, K63, etc.) in the polyubiquitin chains.

• Controlled chain length: The approach enables control over the length of the ubiquitin chains.

• Generation of branched ubiquitin chains: SUE1 can be used to create site-specific branched ubiquitin chains.

• NEDD8 modification capability: The system can even generate NEDD8-modified proteins.

This approach provides a powerful tool for obtaining ubiquitinated proteins to study the biochemical functions of ubiquitination and has applications in diverse research areas, from structural biology to cellular signaling studies .

What are the best methods for distinguishing between UBE2E1's E3-dependent and E3-independent activities?

To differentiate between UBE2E1's E3-dependent and E3-independent activities, researchers can employ several strategic approaches:

• Comparative in vitro ubiquitination assays: Perform parallel reactions with and without known E3 ligase partners of UBE2E1 (such as MuRF1 or RNF34) and compare ubiquitination patterns.

• Structure-guided mutagenesis: Based on the UBE2E1-SETDB1 peptide complex structure, create UBE2E1 mutants that specifically disrupt either E3 binding interfaces or substrate-binding regions involved in E3-independent activity.

• Peptide competition assays: Use SETDB1-derived peptides that can compete for UBE2E1 binding to selectively inhibit E3-independent activity without affecting E3-dependent functions.

• Mass spectrometry analysis of ubiquitin linkages: Different activities of UBE2E1 may generate distinct ubiquitin chain topologies that can be distinguished through detailed mass spectrometry analysis.

• Cell-based assays with E3 ligase knockdowns: Assess UBE2E1 activity in cellular contexts where specific E3 ligases have been knocked down to determine which activities persist in the absence of particular E3 partners.

How does UBE2E1 contribute to ciliopathy pathogenesis?

UBE2E1 plays a significant role in ciliopathy pathogenesis through its functional interaction with MKS1, a protein whose mutations cause Meckel syndrome, a severe ciliopathy. Research findings indicate that:

• UBE2E1 and MKS1 colocalize at the ciliary base .

• Loss of UBE2E1 recapitulates the ciliary phenotypes and aberrant Wnt signaling observed in MKS1 deficiency .

• UBE2E1 mediates ubiquitination of MKS1, affecting both its regulation and degradation .

• The UBE2E1-MKS1 interaction is crucial for processing phosphorylated β-catenin at the ciliary base, thereby regulating canonical Wnt signaling .

These findings provide a mechanistic link between ubiquitin processing at the primary cilium and the regulation of Wnt signaling, a pathway often dysregulated in ciliopathies. The disruption of this process when either UBE2E1 or MKS1 is compromised explains why ciliary dysfunction leads to increased canonical Wnt signaling, a long-recognized but previously mechanistically unclear association .

What is the interactome of UBE2E1 and how does it compare to other class III E2 enzymes?

UBE2E1 participates in a complex protein interaction network that distinguishes it from other class III E2 enzymes (UBE2E2 and UBE2E3). Key interactions include:

Table 2: UBE2E1 Protein Interactions and Their Functional Significance

While all human class III E2s (UBE2E1, UBE2E2, and UBE2E3) can engage with CUL4a complex components , UBE2E1 has specific interactions (such as with MKS1 and RNF34) that may not be shared by other class III E2s. Additionally, UBE2E1's E3-independent ubiquitination capability represents a potentially unique feature among these enzymes .

Complete interactome analysis of UBE2E1 has been undertaken as part of larger studies on the human E2 ubiquitin conjugating enzyme protein interaction network , though comprehensive comparative analysis between all class III E2s remains an area for further research.

How is UBE2E1 activity regulated at the post-translational level?

Post-translational regulation of UBE2E1 represents an important aspect of its functional control, though research in this area is still developing. Based on current understanding, several regulatory mechanisms may influence UBE2E1 activity:

• Co-dependent protein stability: Research indicates that levels of UBE2E1 and MKS1 are co-dependent , suggesting reciprocal regulation of protein stability between these interacting partners.

• Subcellular localization: UBE2E1 shows differential expression between the cytoplasm of slow-twitch muscle fibers and other cellular contexts , indicating localization-dependent regulation mechanisms.

• Auto-ubiquitination: As an E2 enzyme, UBE2E1 may be subject to auto-ubiquitination, a common regulatory mechanism for ubiquitin system enzymes.

• Substrate-induced conformational changes: The structure of UBE2E1 in complex with SETDB1-derived peptide suggests that substrate binding may induce conformational changes that regulate enzymatic activity .

Further research is needed to fully elucidate the post-translational modifications directly affecting UBE2E1 activity, stability, and localization, such as potential phosphorylation, acetylation, or other modifications.

What animal models are most suitable for studying UBE2E1 function in vivo?

Several animal models have proven valuable for studying UBE2E1 function in vivo:

• C57BL/6 mouse models: These have been used to study UBE2E1's role in dexamethasone-induced muscle atrophy. The experimental approach involved treating mice with different dexamethasone doses (1 or 5 mg/kg/day) for varying periods (5, 9, or 14 days) .

• In vivo electroporation models: This technique has been employed for targeted knockdown of UBE2E1 in specific muscles through delivery of miR RNAi constructs. Statistical analysis using both one-way and three-way ANOVA was used to distinguish specific UBE2E1 knockdown effects from transfection and leg effects .

• Mks1-deficient mouse models: Though not directly targeting UBE2E1, these models have been valuable for studying the functional interaction between MKS1 and UBE2E1 in ciliary and Wnt signaling contexts .

When selecting an animal model, researchers should consider the specific aspect of UBE2E1 function they aim to study, whether it's muscle homeostasis, ciliary function, or other cellular processes.

How might UBE2E1-targeted therapies be developed for ciliopathies or muscle disorders?

The involvement of UBE2E1 in both ciliary function and muscle homeostasis suggests potential therapeutic applications in related disorders:

For ciliopathies:

• Small molecules that enhance or stabilize the UBE2E1-MKS1 interaction could potentially compensate for partial loss of MKS1 function in some ciliopathies.

• Targeted delivery of engineered UBE2E1 variants with enhanced β-catenin processing capability could help normalize dysregulated Wnt signaling in ciliary disorders.

For muscle disorders:

• Modulation of UBE2E1 activity could potentially protect against dexamethasone-induced muscle atrophy, which is clinically relevant for patients requiring long-term glucocorticoid treatment .

• Targeted enhancement of UBE2E1 expression or activity in slow-twitch muscle fibers might provide protection against certain forms of muscle atrophy.

Development of such therapies would require:

• High-throughput screening methods to identify compounds that selectively modulate UBE2E1 activity

• Advanced delivery systems for tissue-specific targeting

• Careful assessment of potential off-target effects due to UBE2E1's involvement in multiple cellular pathways

What are the implications of UBE2E1's E3-independent activity for understanding evolutionary aspects of the ubiquitin system?

The discovery of UBE2E1's ability to perform E3-independent ubiquitination raises important questions about the evolution of the ubiquitin system:

• This capability may represent a more primitive mechanism of ubiquitination that predates the evolution of the complex E1-E2-E3 cascade.

• The sequence-dependent nature of UBE2E1's E3-independent activity suggests that specific substrate recognition motifs might have been a precursor to the more elaborate substrate recognition mechanisms mediated by the diverse E3 ligase family.

• Understanding this mechanism could provide insights into how the current three-enzyme ubiquitination cascade evolved and diversified across eukaryotes.

• The structural basis of UBE2E1's E3-independent activity might reveal conserved features that could be present in other E2 enzymes but have been overlooked due to the predominant focus on E3-dependent mechanisms.

This area represents a fascinating frontier in ubiquitin research that bridges biochemistry, structural biology, and evolutionary biology.