UBE2D3 Human

What is UBE2D3 and what is its fundamental role in the ubiquitin-proteasome system?

UBE2D3 is an E2 ubiquitin-conjugating enzyme that functions as an essential intermediary in the ubiquitination cascade. The ubiquitination process involves three classes of enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes like UBE2D3), and E3 (ubiquitin-protein ligases). UBE2D3 receives activated ubiquitin from E1 enzymes and, in cooperation with E3 ligases, transfers ubiquitin to substrate proteins .

For investigating UBE2D3's role in the UPS, researchers should employ:

• In vitro ubiquitination assays using recombinant UBE2D3 protein

• Knockdown experiments using siRNA or shRNA targeting UBE2D3

• Co-immunoprecipitation to identify E3 ligase partners

• Proximity ligation assays (PLA) to measure interactions between UBE2D3 and E3 ligases such as CBL

Research findings indicate that UBE2D3 is one of the most promiscuous E2 enzymes in vitro, functioning with almost every E3 ligase in ubiquitination reactions .

What are the known human substrate proteins of UBE2D3?

Based on current research, several substrates of UBE2D3 have been identified through various methodological approaches:

Methodological approaches to identify UBE2D3 substrates include:

• SILAC-based quantitative diGly-proteomics combined with UBE2D3 depletion

• Label-free diGly proteomics to detect changes in protein ubiquitination

• TULIP2 (Targets of Ubiquitin Ligases Identified by Proteomics 2) methodology to confirm direct targets

• Validation using recombinant proteins in reconstituted ubiquitination assays

These approaches have revealed that UBE2D3 affects both the proteome and the ubiquitinome, with roles in metabolic pathways, cell adhesion, cell signaling, mRNA translation, and protein quality control .

What techniques are commonly used to study UBE2D3 function in vitro?

Several techniques are employed to study UBE2D3 function in vitro:

• Recombinant protein expression and purification:

  • Expression in E. coli as described in search result

  • Typical purification using 50mM HEPES, 150mM NaCl, 2mM DTT, 10% glycerol, pH 7.5 buffer

• In vitro ubiquitination assays with a typical experimental setup:

  • Recombinant E1 enzyme (1-2 μM)

  • Recombinant UBE2D3 (5-10 μM)

  • Recombinant E3 ligase (1-5 μM)

  • Substrate protein (1-10 μM)

  • Ubiquitin (50-100 μM)

  • ATP (2-5 mM) and MgCl₂ (5-10 mM)

  • Analysis by SDS-PAGE and Western blotting

• Binding assays:

  • Proximity ligation assays to measure interactions between UBE2D3 and E3 ligases

  • Co-immunoprecipitation under different cellular conditions

  • Surface plasmon resonance to measure binding kinetics

• Enzymatic activity assays:

  • Measuring ubiquitin transfer from UBE2D3 to substrates

  • Monitoring ATP consumption during ubiquitination reactions

What is the role of UBE2D3 in oocyte meiotic maturation and fertility?

UBE2D3 plays a critical role in oocyte meiotic maturation, with significant implications for fertility research:

• UBE2D3 has been identified as the most highly expressed E2 enzyme in mouse oocytes and is essential for proper meiotic division .

• Experimental findings related to UBE2D3 manipulation:

  • UBE2D3 depletion causes metaphase I (MI) arrest and Cyclin B1 accumulation

  • UBE2D3 overexpression leads to reduced Cyclin B1 levels, kinetochore-microtubule (K-MT) mis-attachments, spindle assembly checkpoint (SAC) dysfunction, and increased aneuploidy

• Age-related fertility implications:

  • UBE2D3 upregulation occurs in oocytes from aged mice

  • This upregulation contributes to age-related meiotic defects

  • These defects can be partially reversed by UBE2D3 knockdown or Cyclin B1 overexpression

• Research methodologies for investigating UBE2D3 in human fertility:

  • RNA interference in human oocytes to assess conservation of function

  • Analysis of UBE2D3 expression in human oocytes of different ages

  • Correlation of UBE2D3 levels with clinical IVF outcomes

This research underscores the importance of the UBE2D3-Cyclin B1 axis in maintaining meiotic fidelity and highlights its potential as a therapeutic target for improving oocyte quality and fertility in aged females .

How does UBE2D3 contribute to protein quality control systems in human cells?

UBE2D3 plays significant roles in protein quality control (PQC) pathways:

• Ribosome-associated quality control (RQC):

  • UBE2D3 is required for the ubiquitination of ribosomal proteins RPS10 and RPS20 by the E3 ligase ZNF598

  • This ubiquitination is important for functional RQC

  • The catalytic activity of UBE2D3 has been shown to be required for RPS10 ubiquitination

• Autophagic protein quality control:

  • UBE2D3 acts at multiple levels in autophagic processes

  • It affects the ubiquitination of SQSTM1 (p62), an important autophagy receptor

• General proteostasis:

  • UBE2D3 depletion affects the proteome and ubiquitinome

  • It impacts proteins involved in various metabolic pathways and cellular processes

Methodological approaches to study UBE2D3's role in PQC include:

• Combination of UBE2D3 depletion with quantitative diGly-based ubiquitinome profiling

• Polysome profiling to assess changes in translation

• Monitoring autophagy flux using LC3 conversion and p62 degradation assays

How does UBE2D3 interact with different E3 ligases, and what determines these interactions?

Understanding UBE2D3-E3 ligase interactions is crucial for deciphering specificity in the ubiquitination system:

• E3 ligase interactions observed in research:

  • Interaction with CBL E3 ligase, which catalyzes ubiquitination of PDFGRα and FGFR1

  • Association with ZNF598 for ribosomal protein ubiquitination

  • Cooperation with RING1B for H2AK119 ubiquitination

• Factors affecting UBE2D3-E3 interactions:

  • Post-translational modifications: The UBE2D3-Ser138Ala mutation results in increased interaction with CBL, suggesting phosphorylation may regulate E3 interactions

  • Protein levels: Increased UBE2D3 levels in the Ser138Ala mutant correlate with stronger CBL interaction

  • Substrate availability: The presence of substrate proteins may enhance or stabilize UBE2D3-E3 interactions

• Methodological approaches for studying these interactions:

  • Proximity ligation assay (PLA) to measure interactions between UBE2D3 and E3 ligases in situ

  • Structure-function analysis using mutational studies

  • In vitro binding assays with purified components

  • Computational modeling of interaction interfaces

What methodologies are most effective for identifying the complete set of in vivo UBE2D3 substrates?

Based on current research, the most effective methodologies for identifying in vivo UBE2D3 substrates include:

• Quantitative diGly proteomics approaches:

  • SILAC-based diGly proteomics with UBE2D3 depletion

  • Label-free diGly proteomics

  • Experimental design: Six biological replicates, all in forward fashion, after confirming the reduction in UBE2D3 protein levels in heavy-labeled samples

• TULIP2 (Targets of Ubiquitin Ligases Identified by Proteomics 2) methodology:

  • This approach confirms substrates as direct targets of UBE2D3

  • Successfully used to identify RPS10, RPS20, and SQSTM1 as direct UBE2D3 targets

• Catalytic activity-dependent validation:

  • Using catalytically inactive UBE2D3 mutants to confirm enzyme-dependent ubiquitination

  • Demonstrating that substrate ubiquitination requires UBE2D3's catalytic activity

• Standard experimental workflow:

  Step	Method	Details
  1	Generate UBE2D3-depleted cells	shRNA knockdown or CRISPR-Cas9 knockout
  2	SILAC labeling	Heavy-labeled control, light-labeled UBE2D3-depleted
  3	Enrichment	Anti-diGly antibodies for ubiquitinated peptides
  4	Analysis	LC-MS/MS mass spectrometry
  5	Identification	Proteins with decreased ubiquitination in UBE2D3-depleted cells
  6	Validation	In vitro ubiquitination assays with candidate proteins
  7	Confirmation	TULIP2 methodology to verify direct targeting

This combined approach provides a powerful tool for identifying in vivo E2 substrates, as demonstrated by the successful identification of multiple UBE2D3 targets .

How can researchers differentiate between the functions of UBE2D3 and other members of the UBE2D family?

The UBE2D family includes several members with high sequence similarity, making functional differentiation challenging:

• Gene-specific targeting strategies:

  • Design of siRNAs or shRNAs that specifically target UBE2D3 "and no other member of the UBE2D family"

  • CRISPR-Cas9 knockout with validation of specificity

  • Complementation studies using UBE2D3-specific constructs

• Protein-specific approaches:

  • Development of antibodies specific to UBE2D3

  • Mass spectrometry-based approaches to identify UBE2D3-specific peptides

  • Expression of tagged versions of UBE2D3 for unambiguous identification

• Evolutionary and structural analyses:

  • Comparative sequence analysis to identify unique regions in UBE2D3

  • Phylogenetic methods to analyze evolutionary relationships between UPS components

  • Profile-based homology detection techniques as described in search result

• Experimental design considerations:

  • Include appropriate controls for specificity

  • Perform rescue experiments with individual UBE2D family members

  • Analyze expression patterns across different cell types and conditions

• Comparative functional analysis:

  • Side-by-side comparison of catalytic activities

  • E3 ligase binding preferences

  • Substrate specificity profiles

These approaches allow researchers to confidently attribute specific functions to UBE2D3 rather than other family members, as demonstrated in the UBE2D3-specific studies described in the search results .