Recombinant Drosophila willistoni Ubiquitin-conjugating enzyme E2 S (GK16201)

What is the molecular structure and key domains of Drosophila willistoni Ubiquitin-conjugating enzyme E2 S?

Drosophila willistoni Ubiquitin-conjugating enzyme E2 S belongs to the class I E2 ubiquitin-conjugating enzyme family. Class I E2s consist primarily of the 16-kD ubiquitin-conjugating (UBC) catalytic domain, which contains a critical catalytic cysteine residue essential for ubiquitin transfer activity . This highly conserved catalytic domain is responsible for interactions with E1 activating enzymes during the ubiquitination cascade. Unlike class II, III, and IV E2 enzymes that possess extensions at their N-terminus, C-terminus, or both termini respectively, class I E2s like the one found in D. willistoni contain only the core UBC domain . The enzyme's structure facilitates its central role in ubiquitin chain assembly, making it not merely a ubiquitin carrier but a key mediator in the ubiquitination process.

How does recombinant Drosophila willistoni Ubiquitin-conjugating enzyme E2 S differ from similar enzymes in related species?

While maintaining high sequence conservation among E2 family members, D. willistoni Ubiquitin-conjugating enzyme E2 S exhibits species-specific variations that reflect evolutionary divergence within the Drosophila genus. Electrophoretic studies of structural genes in the Drosophila willistoni group have demonstrated that despite morphological similarities between sibling species, there exists remarkable genetic differentiation at the molecular level . Research indicates that individuals belonging to different species within the willistoni group differ in approximately half of their gene loci, suggesting substantial genetic diversity despite phenotypic similarity . These distinctions likely extend to subtle differences in the ubiquitin-conjugating enzymes' structures and functions, potentially influencing species-specific proteostasis mechanisms and cellular responses to environmental stressors.

What are the optimal storage and handling conditions for recombinant D. willistoni Ubiquitin-conjugating enzyme E2 S?

For optimal preservation of enzymatic activity, recombinant D. willistoni Ubiquitin-conjugating enzyme E2 S should be stored at -20°C, with extended storage recommended at either -20°C or -80°C . Reconstitution should be performed in deionized sterile water to achieve a protein concentration between 0.1-1.0 mg/mL, followed by the addition of glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage . For working aliquots, storage at 4°C for up to one week is acceptable, but repeated freeze-thaw cycles should be strictly avoided as they significantly compromise enzyme stability and activity . The shelf life of the liquid form is approximately 6 months when stored at -20°C/-80°C, while the lyophilized form can remain stable for up to 12 months under the same storage conditions .

What are the recommended protocols for in vitro ubiquitination assays using D. willistoni Ubiquitin-conjugating enzyme E2 S?

In vitro ubiquitination assays using D. willistoni Ubiquitin-conjugating enzyme E2 S require a carefully optimized reaction system comprising:

Basic Ubiquitination Reaction Components:

The assay should be conducted at 25°C for Drosophila enzymes, as this temperature better reflects the physiological conditions of the organism. Reaction times typically range from 30 minutes to 2 hours, with samples collected at defined intervals to track ubiquitination progression. Analysis is best performed using SDS-PAGE followed by western blotting with anti-ubiquitin antibodies to visualize the formation of mono- and poly-ubiquitinated products. For quantitative analysis, densitometric measurement of ubiquitinated substrate bands can be employed to determine reaction kinetics and enzyme efficiency.

How can researchers effectively assess the enzymatic activity of recombinant D. willistoni Ubiquitin-conjugating enzyme E2 S?

Effective assessment of enzymatic activity requires multiple complementary approaches:

• Thioester Bond Formation Assay: Measure the formation of thioester bonds between the catalytic cysteine residue of E2 S and ubiquitin. This can be detected using non-reducing SDS-PAGE, where the thioester bond is stable under non-reducing conditions but cleaved under reducing conditions.

• Ubiquitin Transfer Assay: Quantify the enzyme's ability to transfer ubiquitin to substrate proteins using radiolabeled or fluorescently labeled ubiquitin. Time-course experiments can determine kinetic parameters including Km and Vmax values.

• In vivo Activity Assessment: Complementation studies in Drosophila models with E2 enzyme deficiencies can demonstrate functional activity. Research on the related E2 enzyme Effete (Eff/UbcD1) has shown its involvement in multiple cellular processes including eye development, germline stem cell maintenance, and apoptosis regulation, providing reference models for such studies .

• Chain-type Specificity Analysis: Determine the enzyme's preference for building specific ubiquitin chain linkages (K48, K63, etc.) using mass spectrometry analysis of ubiquitinated products, as different chain types direct substrates to different cellular fates.

What techniques are most effective for analyzing D. willistoni E2 S interactions with different E3 ligases?

To analyze E2-E3 interactions effectively, researchers should employ a systematic multi-method approach:

• Yeast Two-Hybrid Screening: This technique can identify potential E3 ligase partners from D. willistoni cDNA libraries, providing a foundation for further interaction studies.

• Co-immunoprecipitation (Co-IP): When combined with mass spectrometry, Co-IP can validate and discover novel E2-E3 complexes under physiological conditions. Research on Effete (Eff) has demonstrated its interactions with numerous proteins involved in chromatin organization and telomere protection , suggesting similar complex interaction networks for other E2 enzymes.

• Surface Plasmon Resonance (SPR): SPR provides quantitative binding kinetics data (kon, koff, and KD values) for E2-E3 interactions, enabling researchers to compare binding affinities across different ligase partners.

• Bioluminescence Resonance Energy Transfer (BRET): For real-time monitoring of interactions within living cells, BRET assays can detect dynamic E2-E3 associations under various cellular conditions or treatments.

• NMR Spectroscopy or X-ray Crystallography: These methods can reveal atomic-level details of the binding interface between D. willistoni E2 S and its E3 partners, critical for understanding specificity determinants.

What are the primary cellular functions of D. willistoni Ubiquitin-conjugating enzyme E2 S in proteostasis?

D. willistoni Ubiquitin-conjugating enzyme E2 S plays essential roles in maintaining proteostasis through several interconnected mechanisms:

• Protein Quality Control: Similar to the well-studied UBE2D/Eff enzyme, D. willistoni E2 S likely contributes to the ubiquitination and subsequent degradation of misfolded or damaged proteins . Research has shown that knockdown of specific E2 enzymes can significantly affect the levels of aggregation-prone proteins like huntingtin with expanded polyQ repeats (Htt-polyQ) .

• Regulation of Protein Turnover: The enzyme participates in the targeted degradation of regulatory proteins with short half-lives, enabling rapid cellular responses to changing conditions.

• Stress Response Modulation: E2 enzymes in Drosophila contribute to cellular adaptation to various stressors, similar to the overlapping stress response functions observed in homologous yeast E2 enzymes (UBC1, UBC4, and UBC5) .

• Developmental Regulation: Based on studies of related E2 enzymes like Effete, D. willistoni E2 S likely contributes to specific developmental processes, potentially including eye development and germline stem cell maintenance .

The functional significance of these activities extends beyond basic cellular maintenance to influence organismal development, aging, and stress resistance, positioning E2 enzymes as central regulators of proteome dynamics.

How does D. willistoni E2 S contribute to chromatin organization and gene expression regulation?

Evidence from studies on the related E2 enzyme Effete suggests that D. willistoni E2 S may have significant roles in chromatin organization:

• Chromatin Association: Like Effete, which has been identified as a major component of Drosophila chromatin particularly enriched in repressive chromatin regions , D. willistoni E2 S may directly associate with chromatin structures.

• Telomere Protection: E2 enzymes contribute to telomere integrity, with Effete being required for telomere protection and prevention of telomere fusion . D. willistoni E2 S may serve similar functions in maintaining chromosomal stability.

• Position Effect Variegation Modulation: Effete has been shown to modulate both telomere-induced and heterochromatin-induced position effect variegation (PEV) , suggesting a role in epigenetic regulation. Similar functions may exist for D. willistoni E2 S.

• Histone Modification: Through targeted ubiquitination of histones or chromatin-associated proteins, E2 enzymes contribute to the histone code that regulates gene accessibility and expression.

These chromatin-related functions highlight the diverse roles of E2 enzymes beyond their classical functions in protein degradation pathways, positioning them as multifunctional regulators of nuclear processes.

What phenotypes are associated with altered expression or mutation of Ubiquitin-conjugating enzyme E2 S in Drosophila models?

Altered expression or mutation of E2 enzymes in Drosophila is associated with diverse phenotypes that reflect their multiple cellular functions:

• Proteostasis Disruption: Studies of UBE2D/Eff demonstrate that knockdown can increase levels of aggregation-prone proteins like huntingtin with expanded polyQ repeats, particularly high-molecular-weight aggregates that accumulate in the stacking gel during SDS-PAGE analysis .

• Developmental Abnormalities: Based on Effete studies, disruption of E2 S function may lead to eye development defects and abnormalities in the maintenance of female germline stem cells .

• Apoptotic Dysregulation: E2 enzymes regulate programmed cell death, with Effete known to be involved in apoptosis regulation . Disruption of D. willistoni E2 S may therefore affect normal cell death patterns during development and tissue homeostasis.

• Chromosomal Instability: Given the role of E2 enzymes in telomere protection, altered E2 S expression may lead to telomere fusion events and genomic instability .

• Stress Sensitivity: Similar to yeast models where deletion of multiple E2 genes renders cells sensitive to various stressors , mutation of D. willistoni E2 S may compromise stress resistance mechanisms.

These phenotypes collectively illustrate the essential nature of E2 enzymes in maintaining cellular and organismal homeostasis across multiple biological processes.

How does D. willistoni E2 S compare structurally and functionally to homologous enzymes in model organisms?

Comparative analysis reveals both conservation and divergence among E2 enzymes across species:

The D. willistoni E2 S preserves the core catalytic mechanisms of the E2 enzyme family while potentially incorporating species-specific adaptations. Studies of the Drosophila willistoni group have revealed that despite morphological similarities between sibling species, there exists significant genetic differentiation, with individuals from different species differing in approximately half of their gene loci . This genetic divergence likely contributes to subtle functional specializations of E2 enzymes across Drosophila species, potentially adapting them to different ecological niches and environmental challenges.

What evolutionary insights can be gained from studying D. willistoni E2 S in relation to the willistoni species group?

Studying D. willistoni E2 S offers valuable evolutionary insights:

These evolutionary perspectives contribute to our understanding of how essential cellular mechanisms diversify during speciation while maintaining core functionalities necessary for organismal survival.

What techniques are most informative for phylogenetic analysis of E2 enzymes across Drosophila species?

Several complementary approaches provide robust phylogenetic insights:

• Sequence-Based Phylogenetics: Maximum likelihood and Bayesian inference methods applied to aligned E2 sequences can reconstruct evolutionary relationships. Focus on both conserved catalytic domains and variable regions provides a comprehensive evolutionary picture.

• Structural Phylogenetics: Comparing predicted three-dimensional structures of E2 enzymes can reveal functional clustering that may not be apparent from sequence data alone.

• Expression Pattern Analysis: Examining when and where E2 enzymes are expressed across species can reveal evolutionary shifts in regulation that may contribute to functional divergence.

• Interactome Comparison: Characterizing E2-E3 interaction networks across species can identify evolutionary changes in protein-protein interaction patterns that may drive functional specialization.

• Gel Electrophoresis for Polymorphism Detection: As demonstrated in studies of the Drosophila willistoni group, gel electrophoresis remains valuable for detecting structural gene variants across populations and species , particularly when combined with modern molecular techniques.

These approaches collectively provide a multi-dimensional view of E2 enzyme evolution, revealing patterns of conservation and divergence that illuminate both the core functions and species-specific adaptations of these essential proteins.

How can D. willistoni E2 S be utilized in investigating age-related proteostasis decline?

D. willistoni E2 S offers a powerful tool for studying age-related proteostasis mechanisms:

• Model System Approach: Drosophila provides an excellent model for aging studies due to its relatively short lifespan and genetic tractability. Research has shown that UBE2D/Eff maintains a youthful proteostasis network in Drosophila , suggesting D. willistoni E2 S may have similar roles in age-related protein quality control.

• Aggregation Assays: Using model aggregation-prone proteins like huntingtin-polyQ expressed in Drosophila tissues, researchers can assess how modulation of E2 S levels affects aggregate formation throughout aging . This approach has revealed that knockdown of certain E2 enzymes can significantly impact the accumulation of high-molecular-weight protein aggregates.

• Tissue-Specific Analysis: By examining E2 enzyme activity in different tissues throughout the aging process, researchers can identify tissue-specific vulnerabilities to proteostasis decline. The Drosophila retina has proven particularly useful for such studies .

• Comparative Longevity Studies: Manipulating E2 S expression levels in D. willistoni and examining effects on lifespan and healthspan can reveal connections between ubiquitination efficiency and aging outcomes.

• Stress Response Integration: Examining how age affects E2 S-mediated responses to various stressors can illuminate the molecular basis of declining stress resistance in aging organisms.

These approaches contribute to understanding how ubiquitination pathways influence the aging process and may identify intervention points for preserving proteostasis during aging.

What methodological innovations enable identification of specific substrates for D. willistoni E2 S?

Several cutting-edge approaches facilitate substrate identification:

• Proximity-Dependent Biotin Identification (BioID): By fusing E2 S to a biotin ligase, researchers can biotinylate and subsequently identify proteins that come into close proximity with the enzyme in vivo, revealing potential substrates and interaction partners.

• Ubiquitin Remnant Profiling: Using antibodies that recognize the di-glycine remnant left on lysine residues after tryptic digestion of ubiquitinated proteins, combined with mass spectrometry, researchers can identify ubiquitination sites proteome-wide following E2 S manipulation.

• Tandem Ubiquitin Binding Entities (TUBEs): These engineered proteins with high affinity for poly-ubiquitin chains can enrich ubiquitinated proteins from cell lysates for subsequent identification by mass spectrometry, allowing comparison between wild-type and E2 S-manipulated conditions.

• Orthogonal Ubiquitin Transfer (OUT): This approach uses engineered ubiquitin and E2 enzyme pairs that function orthogonally to the endogenous ubiquitination machinery, enabling specific labeling and identification of direct E2 substrates.

• Global Protein Stability Profiling: By combining protein synthesis inhibition with quantitative proteomics, researchers can identify proteins whose stability depends on E2 S activity, indicating potential substrates.

These methodologies collectively enhance our ability to decipher the complex substrate networks of E2 enzymes, moving beyond candidate approaches to systematic identification of ubiquitination targets.

How can computational modeling enhance understanding of D. willistoni E2 S catalytic mechanisms?

Computational approaches provide valuable insights into E2 enzyme function:

• Molecular Dynamics Simulations: These can model the conformational changes that occur during the ubiquitin transfer reaction, revealing transient states and energy barriers that are difficult to capture experimentally.

• Quantum Mechanics/Molecular Mechanics (QM/MM): For studying the chemical reaction mechanism of ubiquitin transfer, QM/MM can model bond breaking and formation at the catalytic cysteine while accounting for the protein environment.

• Protein-Protein Docking: In silico docking studies can predict binding modes between D. willistoni E2 S and various E3 ligases or substrate proteins, identifying key interaction surfaces and specificity determinants.

• Systems Biology Modeling: Network-based approaches can integrate E2 enzymes into larger ubiquitination pathway models, predicting system-level responses to perturbations in E2 activity.

• Evolutionary Coupling Analysis: By analyzing patterns of co-evolution between amino acid positions, researchers can identify functionally important residues and interaction surfaces that have been conserved throughout evolution.

These computational approaches complement experimental studies by providing mechanistic insights at atomic resolution and system-level perspectives that are challenging to obtain through experimental means alone.

What are common challenges in expressing and purifying recombinant D. willistoni E2 S and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant E2 enzymes:

• Solubility Issues: E2 enzymes may form inclusion bodies during bacterial expression.

  • Solution: Optimize expression conditions by lowering temperature (16-18°C), using specialized expression strains (Rosetta, Arctic Express), or adding solubility tags (SUMO, MBP).

• Catalytic Activity Preservation: The critical catalytic cysteine is prone to oxidation during purification.

  • Solution: Include reducing agents (DTT or TCEP) throughout the purification process and consider performing purification under anaerobic conditions for sensitive applications.

• Thioester Bond Stability: The thioester bond formed between E2 and ubiquitin is labile.

  • Solution: For activity assays, prepare samples immediately before use and maintain them at appropriate pH (typically 7.5-8.0) to preserve thioester linkages.

• Aggregation During Storage: Purified E2 enzymes may aggregate during storage.

  • Solution: As recommended for similar recombinant proteins, store at -20°C or -80°C with 5-50% glycerol, avoid repeated freeze-thaw cycles, and maintain working aliquots at 4°C for no more than one week .

• Expression System Selection: The source organism affects post-translational modifications.

  • Solution: While E. coli is suitable for basic structural studies, consider yeast or insect cell expression systems for functional studies requiring native-like modifications.

How can researchers troubleshoot inconsistent results in ubiquitination assays using D. willistoni E2 S?

When facing inconsistent ubiquitination assay results, systematic troubleshooting is essential:

• Enzyme Activity Verification: Conduct thioester loading assays before complex ubiquitination reactions to ensure E2 S is catalytically active.

• Component Quality Control: Establish quality control measures for all reaction components:

  • Test ubiquitin for proper folding using circular dichroism

  • Verify ATP quality with a luciferase assay

  • Confirm E1 activity with a pyrophosphate release assay

• Buffer Optimization: Ionic strength, pH, and reducing agent concentration significantly impact ubiquitination reactions:

  • Titrate buffer components across multiple reactions

  • Consider the impact of salt concentration on protein-protein interactions

  • Ensure reducing conditions are maintained throughout the reaction

• Temperature Control: Maintain consistent temperature during reactions, as enzymatic rates are highly temperature-dependent.

• Time Course Analysis: Rather than endpoint measurements, conduct time course experiments to capture reaction kinetics and identify optimal sampling points.

• Detergent Considerations: Low concentrations of non-ionic detergents (0.01-0.05% Triton X-100) can reduce non-specific protein binding to tubes and improve consistency.

• Detection Method Validation: When using antibody-based detection of ubiquitination, validate antibody specificity and consider alternative detection methods for confirmation.

What are the key considerations for designing CRISPR/Cas9-mediated gene editing experiments targeting endogenous D. willistoni E2 S?

CRISPR/Cas9 targeting of E2 enzymes requires careful experimental design:

• Guide RNA Selection: E2 enzymes contain highly conserved regions that may create off-target effects.

  • Strategy: Use algorithms that account for genome-wide off-target predictions, prioritizing guides with at least 3-4 mismatches to any other genomic region.

  • Validation: Sequence verify multiple potential off-target sites after editing.

• Functional Domain Preservation: The catalytic cysteine and surrounding residues are critical for function.

  • Strategy: Design edits that avoid disrupting the catalytic core; target less conserved regions for insertions or modifications.

  • Validation: Confirm enzyme activity after editing using activity assays.

• Phenotypic Consequences: E2 enzymes often have essential functions.

  • Strategy: Consider conditional approaches (tissue-specific or inducible systems) rather than complete knockouts, which may be lethal.

  • Validation: Assess developmental timing and viability in edited lines.

• Redundancy Considerations: Multiple E2 enzymes may have overlapping functions.

  • Strategy: Consider multiplex editing to target functionally redundant E2 enzymes simultaneously.

  • Validation: Perform complementation studies with related E2 enzymes.

• Editing Efficiency Verification: Confirm successful editing at the genomic, transcript, and protein levels.

  • Strategy: Design PCR primers for genomic verification, RT-PCR for transcript analysis, and develop specific antibodies or tagged constructs for protein detection.

  • Validation: Sequence the edited region and quantify editing efficiency.