UBE2Z Human

What is UBE2Z and what is its primary function in human cells?

UBE2Z, also known as HOYS7, Use1, or Uba6-specific E2 conjugating enzyme 1, functions as a ubiquitin-conjugating enzyme (E2) that catalyzes the covalent attachment of ubiquitin to target proteins. It acts as a specific substrate for UBA6 and is not charged with ubiquitin by UBE1, which distinguishes it from most other E2 enzymes . UBE2Z is classified as a class IV E2 enzyme based on its domain organization, containing both N- and C-terminal extensions flanking the conserved UBC core domain . Beyond ubiquitination, UBE2Z plays a critical role in the FAT10 conjugation pathway, a post-translational modification system analogous to ubiquitination. Current evidence suggests that UBE2Z may also be involved in apoptosis regulation, though the exact mechanisms require further investigation .

How is UBE2Z structurally characterized and how does this relate to function?

The crystal structure of UBE2Z has been determined at 2.1Å resolution, providing significant insights into its functional architecture. UBE2Z is a 354-residue protein comprising a conserved core UBC domain with approximately 100-residue N- and C-terminal extensions . The core domain shares about 34% sequence identity with UBE2D3 (UBCH5C) . While full-length UBE2Z was used in crystallization trials, the solved structure lacks the N-terminal 98 residues, suggesting this region may be intrinsically disordered or highly flexible .

The structural data reveals that UBE2Z possesses unique features that distinguish it from typical E2 enzymes:

The structural information helps explain how UBE2Z achieves specificity in the FAT10 conjugation pathway while maintaining its ability to function in ubiquitination .

What are the known protein interactions of UBE2Z?

UBE2Z exhibits specific interactions that determine its role in ubiquitin and FAT10 conjugation pathways:

• UBA6 Interaction: UBE2Z is specifically activated by UBA6 (E1) but not by UBE1, the primary ubiquitin-activating enzyme. This specificity is mediated by structural features that enable selective recognition .

• FAT10 Interaction: UBE2Z interacts with FAT10, a ubiquitin-like protein, facilitating its conjugation to target proteins. The C-terminal CYCI tetrapeptide of FAT10 is critical for specificity toward UBA6 and UBE2Z .

• E3 Ligase Interactions: While the search results don't specifically identify E3 ligases that work with UBE2Z, as an E2 enzyme, UBE2Z is expected to interact with one or more E3 ligases to complete the ubiquitin/FAT10 transfer to substrate proteins.

The specific LB loop region and the N-terminal extension in UBE2Z have been shown to be essential for selectivity toward UBA6, highlighting the structural basis for these protein interactions .

What are the recommended expression systems and purification strategies for recombinant UBE2Z?

Based on the search results, the following expression and purification approach has been successfully employed for UBE2Z:

Expression System:

• Host: Escherichia coli

• Induction: 0.1 mM isopropyl 1-thio-β-d-galactopyranoside

• Growth Conditions: Overnight at 15°C (important for proper folding)

Purification Protocol:

• Initial purification using Talon beads (metal affinity chromatography) in buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 1 mM TCEP, and 10% (w/v) glycerol

• Elution with 250 mM imidazole (pH 7.5)

• Overnight dialysis at 4°C against 20 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, and 10% (w/v) glycerol with HRV 3C protease to remove His6 tags

• Further purification using ResourceQ column (anion exchange chromatography) with a gradient to 1 M NaCl

This protocol has yielded UBE2Z with >95% purity and <1 EU/μg endotoxin level, suitable for various analytical techniques including SDS-PAGE and HPLC .

What methods can be used to assess UBE2Z enzymatic activity in vitro?

Several methods have been documented for assessing UBE2Z activity:

Thioester Formation Assay:
This assay measures the formation of thioester bonds between UBE2Z and ubiquitin or FAT10:

• Prepare reaction mixture containing:

  • 100 nM UBA6 (E1 enzyme)

  • 2.5 μM Cyanine5-labeled ubiquitin or FAT10

  • 2 mM ATP

  • Varying concentrations of UBE2Z (0-32 μM)

  • Buffer: 20 mM HEPES (pH 7.5), 150 mM NaCl, 10% (w/v) glycerol

• Incubate for appropriate time (45 seconds for ubiquitin or FAT10 LRLR variant; 4 minutes for wild-type FAT10 or Ub CYCI variant)

• Stop reaction with non-reducing SDS-PAGE buffer

• Separate products on 4-12% NuPAGE gels in MES buffer

• Quantify E2~UBL bands using fluorescence imaging

Verification of Thioester Bond:
To confirm that the observed E2~UBL bands represent genuine thioester conjugates (rather than non-covalent complexes or isopeptide bonds), include a control with 5 mM β-mercaptoethanol, which should reduce the thioester bond .

Kinetic Analysis:
Initial rates at different E2 concentrations can be fitted using non-linear regression to the Michaelis-Menten model or to a substrate inhibition effect equation to determine apparent Vmax and Km values .

How can researchers study the structural features of UBE2Z?

Several structural biology approaches have been successfully applied to UBE2Z:

X-ray Crystallography:
The structure of UBE2Z has been solved at 2.1Å resolution using the following approach:

• Crystallization in space group P22121 with cell dimensions a=45.33Å, b=57.81Å, c=105.11Å

• Data collection at synchrotron radiation source (ESRF)

• Molecular replacement using UBE2D3 (PDB code 1X23) as a search model

• Model improvement using phenix.mr_rosetta

• Experimental phasing with crystals soaked in methylmercuric chloride

• Iterative refinement using REFMAC or BUSTER, alternating with manual building in COOT

Structure Validation:

• Optimize structures using PDB_REDO

• Validate using the Molprobity server

• Assess Ramachandran statistics (97.35% favored residues, 0% outliers in the UBE2Z structure)

Mutagenesis Studies:
Structure-guided mutagenesis can be used to investigate the functional importance of specific residues or regions, particularly:

• The LB loop region

• The N-terminal extension

• The active site cysteine residue

How does UBE2Z differ from other ubiquitin-conjugating enzymes?

UBE2Z exhibits several distinctive features that set it apart from typical E2 enzymes:

Unlike most E2 enzymes that are charged with ubiquitin by UBE1, UBE2Z specifically requires UBA6 for activation. This specificity appears to be governed by distinct recognition mechanisms, potentially similar to those observed between the SUMO E1 Cys domain and the SUMO E2 enzyme UBE2I, or the NEDD8 E2 UBE2M that uses an N-terminal extension to interact with the NEDD8 E1 component UBA3 .

Additionally, while the UBC core domain is common to all E2 enzymes, UBE2Z's N- and C-terminal extensions likely contribute to its distinct functional properties, including its ability to participate in both ubiquitination and FAT10 conjugation pathways .

What is the role of the UBE2Z N-terminal extension and how does it affect function?

The N-terminal extension of UBE2Z, comprising approximately 100 residues, appears to play crucial roles in the enzyme's function:

• E1 Recognition: Experimental data indicate that the N-terminal extension is essential for selectivity toward UBA6, suggesting it mediates specific E1-E2 interactions that are critical for the conjugation pathway .

• Structural Flexibility: Although the full-length UBE2Z was used in crystallization trials, the N-terminal 98 residues were not visible in the crystal structure, suggesting this region may be intrinsically disordered or highly flexible . This flexibility could be important for accommodating different binding partners or conformational changes during the catalytic cycle.

• Evolutionary Conservation: Despite showing poor sequence similarity with N-terminal extensions of other class II or class IV E2 enzymes, this region is well conserved in mammalian UBE2Z orthologs, indicating its functional importance in higher organisms .

• Pathway Specificity: The N-terminal extension likely contributes to the enzyme's ability to function in both ubiquitination and FAT10 conjugation pathways, possibly by mediating distinct interactions with pathway components.

While the precise molecular mechanisms remain to be fully elucidated, these findings highlight the N-terminal extension as a key determinant of UBE2Z's functional specificity and suggest potential avenues for further investigation.

How does the FAT10 C-terminal CYCI tetrapeptide influence UBE2Z activity?

The C-terminal CYCI tetrapeptide of FAT10 plays a critical role in determining specificity in the FAT10 conjugation pathway:

• Specificity Determinant: Experimental analyses have demonstrated that the CYCI tetrapeptide is essential for specificity toward both UBA6 and UBE2Z, distinguishing FAT10 from other ubiquitin-like proteins .

• Transfer Rate Modulation: Surprisingly, the CYCI motif actually slows down transfer rates for FAT10 from UBA6 onto UBE2Z . This finding suggests that the specificity-determining motif introduces a kinetic constraint in the conjugation pathway.

• Mechanistic Implications: The observed slowing effect indicates that the CYCI motif might enforce additional conformational requirements or interaction constraints that ensure pathway fidelity at the expense of reaction speed.

• Experimental Verification: Researchers have demonstrated this effect through comparative kinetic analyses of wild-type FAT10 versus mutant variants, showing significant differences in transfer rates that correlate with the presence of the CYCI motif .

This counterintuitive relationship between specificity and efficiency highlights the complex evolutionary trade-offs in ubiquitin-like conjugation pathways and suggests that precise regulation may sometimes be prioritized over catalytic speed.

What are common challenges in UBE2Z activity assays and how can they be addressed?

Researchers working with UBE2Z may encounter several technical challenges:

• Establishing Appropriate Reaction Times:

  • Challenge: UBE2Z exhibits different reaction kinetics depending on the UBL partner (ubiquitin vs. FAT10) and specific variant.

  • Solution: Conduct preliminary time-course experiments to determine optimal reaction times. Based on published protocols, reactions with ubiquitin or FAT10 LRLR variant should be stopped after 45 seconds, while reactions with wild-type FAT10 or Ub CYCI variant require 4 minutes .

• Distinguishing Thioester from Isopeptide Bonds:

  • Challenge: E2~UBL bands could represent either thioester linkages (the active intermediate) or stable isopeptide bonds.

  • Solution: Include a reducing agent control (e.g., 5 mM β-mercaptoethanol) which should cleave thioester but not isopeptide bonds .

• Selecting Appropriate Kinetic Models:

  • Challenge: UBE2Z reactions may exhibit substrate inhibition at higher concentrations.

  • Solution: When fitting kinetic data, compare fits to both standard Michaelis-Menten and substrate inhibition models to determine which better describes the observed behavior .

• Working with Combined Enzyme Systems:

  • Challenge: UBE2Z loading assays require the combined activity of both UBA6 and UBE2Z, complicating kinetic analyses.

  • Solution: Report apparent Vmax and Km values to reflect the combined activity of the UBA6-UBE2Z couple rather than individual enzyme parameters .

How can researchers ensure the production of functionally active UBE2Z?

Ensuring the production of active UBE2Z requires careful consideration of several factors:

• Expression Conditions:

  • Use low temperature induction (15°C overnight) to promote proper folding

  • Ensure the expression construct preserves all functional domains, including the N- and C-terminal extensions

• Purification Strategy:

  • Employ a two-step purification approach (metal affinity followed by ion exchange chromatography)

  • Maintain reducing conditions (e.g., 1 mM TCEP) throughout purification to protect the active site cysteine

• Quality Control Assessments:

  • Verify protein purity (>95%) using SDS-PAGE and HPLC

  • Confirm proper folding using circular dichroism or thermal shift assays

  • Test enzymatic activity using thioester formation assays with fluorescently labeled ubiquitin

• Storage Considerations:

  • Include glycerol (10% w/v) in storage buffers to maintain stability

  • Store aliquots at -80°C to prevent repeated freeze-thaw cycles

  • Verify activity after storage using established activity assays

What controls should be included in UBE2Z functional studies?

Robust UBE2Z functional studies should incorporate several key controls:

• Catalytic Cysteine Mutant:

  • Generate a UBE2Z variant with the active site cysteine mutated to alanine

  • This should abolish thioester formation and serve as a negative control for enzymatic activity

• E1 Enzyme Controls:

  • Include reactions without UBA6 to confirm E1-dependency

  • Use UBE1 instead of UBA6, which should not charge UBE2Z with ubiquitin, confirming E1 specificity

• UBL Specificity Controls:

  • Compare reactions with ubiquitin versus FAT10

  • Include UBL variants (e.g., FAT10 with the CYCI motif mutated) to validate specificity determinants

• Thioester Bond Verification:

  • Run parallel samples with and without reducing agent to confirm the thioester nature of the E2~UBL linkage

• Time-Course Controls:

  • Verify that measurements are taken within the linear range of the reaction

  • Ensure reaction times are appropriate for the specific UBL being tested (shorter for ubiquitin, longer for FAT10)

• Fluorophore Impact Control:

  • When using fluorescently labeled UBLs, verify that the label does not perturb kinetic measurements by conducting competition experiments between labeled and unlabeled UBLs

How does the crystal structure of UBE2Z advance our understanding of class IV E2 enzymes?

The UBE2Z crystal structure at 2.1Å resolution provides several important insights that advance our understanding of class IV E2 enzymes:

• Domain Organization: The structure reveals how the different domains of UBE2Z are organized, showing the spatial arrangement of the UBC core domain relative to the C-terminal extension (the N-terminal extension was not visible in the structure) .

• Structural Conservation and Divergence: The UBE2Z structure demonstrates both conservation of the UBC core domain (34% sequence identity with UBE2D3) and unique features that distinguish it from other E2 enzymes, providing a molecular basis for its unique functional properties .

• Functional Elements: The structure identifies key regions including the LB loop that are essential for selectivity toward UBA6, advancing our understanding of E1-E2 recognition specificity .

• Comparison with Other Class IV E2s: The UBE2Z structure reveals emerging similarities between class IV E2 enzymes, suggesting common structural principles despite limited sequence conservation in the extension domains .

This structural information provides a foundation for future structure-guided investigations of UBE2Z function and for developing targeted approaches to modulate its activity in experimental or therapeutic contexts.

What are the implications of UBE2Z's dual role in ubiquitin and FAT10 conjugation pathways?

UBE2Z's ability to function in both ubiquitination and FAT10 conjugation has several important implications:

• Pathway Crosstalk: UBE2Z may serve as a regulatory node mediating crosstalk between ubiquitin and FAT10 conjugation pathways, potentially enabling coordinated control of these distinct post-translational modifications.

• Substrate Fate Determination: By participating in both pathways, UBE2Z could direct substrates toward different fates depending on whether they are modified with ubiquitin (often leading to proteasomal degradation) or FAT10 (which may have distinct functional consequences).

• Regulatory Mechanisms: The dual functionality raises questions about how UBE2Z's activity is regulated to direct it toward one pathway versus the other, potentially involving additional factors, post-translational modifications, or compartmentalization.

• Evolutionary Significance: The ability to function with both UBLs suggests UBE2Z may have evolved from a common ancestor of both pathways or represents an evolutionary adaptation to coordinate these related but distinct conjugation systems.

• Redundancy and Robustness: The dual role could provide functional redundancy that ensures critical cellular processes remain operational even if one pathway is compromised.

Understanding the mechanisms governing UBE2Z's participation in these distinct pathways could provide insights into the broader question of how cells coordinate different UBL conjugation systems.

What therapeutic potential might emerge from understanding UBE2Z function?

While the search results don't directly address therapeutic applications, several potential avenues can be inferred from our understanding of UBE2Z:

• Cancer Therapy: Given UBE2Z's potential role in apoptosis regulation , modulating its activity might provide approaches for promoting cancer cell death or overcoming apoptosis resistance.

• Inflammatory Disorders: FAT10 is induced by inflammatory cytokines and has been implicated in inflammation-associated conditions. As a key enzyme in FAT10 conjugation, UBE2Z could represent a target for modulating inflammatory responses.

• Pathway-Specific Modulation: The structural and biochemical understanding of how UBE2Z distinguishes between ubiquitin and FAT10 conjugation could enable the development of molecules that selectively modulate one pathway while sparing the other.

• Diagnostic Applications: Expression levels or activity profiles of UBE2Z might serve as biomarkers for conditions involving dysregulation of ubiquitin or FAT10 conjugation pathways.

• Tool Development: Engineered UBE2Z variants with altered specificity or activity could serve as research tools for investigating the roles of ubiquitin and FAT10 modification in various cellular processes.

Future research focusing on UBE2Z's roles in specific disease contexts will be essential to validate and develop these potential therapeutic applications.