Recombinant Mouse Ubiquitin-conjugating enzyme E2 D2B (Ube2d2b)

What is Recombinant Mouse Ubiquitin-conjugating enzyme E2 D2B (Ube2d2b)?

Recombinant Mouse Ube2d2b is a member of the ubiquitin-conjugating (E2) enzyme family that plays a crucial role in the ubiquitin-proteasome system. This E2 enzyme contains a catalytic core domain with an active site cysteine residue that forms a thioester bond with ubiquitin during the ubiquitination cascade. Ube2d2b functions as an intermediary between E1 (ubiquitin-activating) and E3 (ubiquitin ligase) enzymes in the protein ubiquitination pathway, thereby facilitating protein degradation, quality control, and numerous cellular signaling processes .

What is the molecular structure and weight of Recombinant Mouse Ube2d2b?

Recombinant Mouse Ube2d2b has a molecular weight of approximately 17 kDa, similar to its human ortholog UBE2D2. When charged with ubiquitin, the complex has a combined molecular weight of approximately 25 kDa (17 kDa for the enzyme and 8.6 kDa for ubiquitin) . The protein contains an E2 catalytic core domain that is highly conserved across species. This domain houses the critical active site cysteine residue responsible for forming the thioester bond with ubiquitin before transferring it to substrate proteins in conjunction with E3 ligases .

How conserved is Mouse Ube2d2b compared to human UBE2D2?

Mouse Ube2d2b shares extremely high sequence homology with human UBE2D2, with approximately 100% amino acid sequence identity in the catalytic domain. This remarkable conservation underscores the evolutionary importance of this enzyme in the ubiquitination pathway. The high homology allows for cross-species functional studies, as demonstrated in Drosophila models where human UBE2D2 successfully rescued phenotypes caused by knockdown of the Drosophila homolog effete (eff) . This conservation extends to the active site cysteine and structural elements critical for interaction with E1, E3 enzymes, and ubiquitin.

Where is Ube2d2b primarily expressed and localized in mouse tissues?

Similar to human UBE2D2, mouse Ube2d2b is widely expressed across multiple tissues. It localizes to both the nucleus and cytoplasm, allowing it to participate in ubiquitination events in both cellular compartments . Expression analysis shows consistent presence in skeletal muscle, brain, heart, liver, and other major organs. The broad expression pattern correlates with its fundamental role in cellular protein quality control. Immunohistochemistry and subcellular fractionation studies have confirmed this dual localization pattern, which is essential for its diverse functions in different cellular compartments.

How does Ube2d2b contribute to proteostasis during aging in mice?

Studies of UBE2D homologs provide insights into Ube2d2b's role in aging. In Drosophila, the homolog effete (eff) shows declining protein levels with age in skeletal muscle, and its knockdown causes accelerated accumulation of insoluble poly-ubiquitinated proteins - a hallmark of aging tissues . Experimental evidence indicates that Ube2d2b likely maintains optimal proteasome function throughout the lifespan, as its reduction correlates with decreased proteolytic activity of the proteasome. Mouse models with tissue-specific Ube2d2b knockdown display premature aging phenotypes, suggesting it plays a crucial role in preventing age-related proteostasis decline through maintaining efficient protein quality control mechanisms .

What types of ubiquitin chain linkages does Ube2d2b preferentially catalyze?

Based on studies of human UBE2D2, mouse Ube2d2b is capable of mediating the formation of ubiquitin chains linked through multiple lysine residues, particularly Lys11, Lys48, and Lys63 . This versatility is significant as these different linkage types direct proteins to distinct cellular fates. Lys48-linked chains primarily target proteins for proteasomal degradation, Lys63-linked chains often function in non-degradative signaling pathways, and Lys11-linked chains can be involved in cell cycle regulation. This multi-linkage capability allows Ube2d2b to participate in diverse cellular processes beyond simple protein degradation, including DNA repair, inflammatory responses, and developmental pathways.

How does Ube2d2b activity influence protein aggregation in neurodegenerative disease models?

Research with Drosophila models reveals that the UBE2D family plays differential roles in handling aggregation-prone proteins like polyglutamine-expanded huntingtin (Htt-polyQ). While some E2 enzymes mediate aggregate assembly, UBE2D/effete is specifically required for huntingtin-polyQ degradation . Experiments show that UBE2D2 expression can rescue retinal degeneration induced by effete knockdown in polyglutamine disease models, and this protection results from enhanced degradation of high-molecular-weight Htt-polyQ species . Mouse Ube2d2b likely plays similar protective roles against protein aggregation by facilitating the degradation of misfolded proteins before they form toxic aggregates, suggesting its relevance as a therapeutic target in neurodegenerative disorders.

What is the relationship between Ube2d2b and stress response pathways?

Proteomics analyses of UBE2D-deficient tissues reveal upregulation of several stress response proteins, including chaperones and proteasome components . This suggests that Ube2d2b deficiency triggers compensatory stress responses aimed at restoring proteostasis. The enzyme appears to interface with both the unfolded protein response and heat shock response pathways. When Ube2d2b function is compromised, cells increase expression of stress-responsive genes to compensate for the loss of this critical ubiquitination enzyme. These findings indicate that Ube2d2b helps maintain basal proteostasis, and its decline may contribute to age-related increases in cellular stress and corresponding adaptive responses.

What are the optimal reaction conditions for in vitro ubiquitination assays using Recombinant Mouse Ube2d2b?

For in vitro ubiquitination assays, pre-charged Ube2d2b-ubiquitin complexes can be added directly to reactions containing E3 ubiquitin ligases and substrates, eliminating the need for ATP, E1 enzyme, or additional ubiquitin . Typical reaction buffers contain 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM MgCl₂. Importantly, reducing agents such as dithiothreitol (DTT) or β-mercaptoethanol should be avoided or used at minimal concentrations as they can cause unintended thiolytic release of ubiquitin from the Ube2d2b-ubiquitin complex. Instead, TCEP (tris(2-carboxyethyl)phosphine) is recommended as a thioester-friendly reductant . Reaction temperature is typically maintained at 30°C, and reaction times range from 30 minutes to 2 hours depending on the specific E3 ligase and substrate combination being studied.

How can I design effective knockdown experiments to study Ube2d2b function in mouse models?

When designing knockdown experiments for Ube2d2b, consider tissue-specific approaches to avoid developmental lethality associated with complete knockout. Conditional knockdown using Cre-loxP systems or inducible shRNA/siRNA can provide temporal and spatial control. Based on Drosophila studies, expression of a validated RNAi construct driven by tissue-specific promoters (such as MhcF3-Gal4 for muscle-specific expression) can achieve effective knockdown .
For mouse models, AAV-delivered shRNA constructs or CRISPR-Cas9 with tissue-specific promoters offer good options. Always include appropriate controls including scrambled RNAi sequences and monitor knockdown efficiency through qPCR and western blotting. To validate phenotypes, rescue experiments using RNAi-resistant Ube2d2b constructs or human UBE2D2 (given the high conservation) should be conducted .

What technical considerations are important when working with ubiquitin-charged Ube2d2b protein?

When working with ubiquitin-charged Ube2d2b, several technical considerations are crucial. First, avoid reducing agents like DTT and β-mercaptoethanol that can cleave the thioester bond between Ube2d2b and ubiquitin . Second, store the protein at -80°C and minimize freeze-thaw cycles to preserve the thioester linkage. Third, before use in assays, confirm the integrity of the ubiquitin-charged enzyme by non-reducing SDS-PAGE, which should show a band at approximately 25 kDa (representing the Ube2d2b-ubiquitin complex) .
When designing in vitro reactions, optimize temperature, pH, and salt concentrations for your specific E3 ligase and substrate. Additionally, include appropriate controls such as catalytically inactive Ube2d2b (with the active site cysteine mutated to alanine) to distinguish between enzymatic activity and non-specific effects in your experimental system.

How can I analyze Ube2d2b-dependent changes in the muscle proteome?

To analyze Ube2d2b-dependent changes in the muscle proteome, employ tandem mass tag (TMT)-based proteomics comparing tissues with Ube2d2b knockdown versus control, as well as rescue conditions with human UBE2D2 expression . Sample preparation should include both detergent-soluble and insoluble fractions to capture changes in protein aggregation. Analyze data for enriched protein categories among significantly regulated proteins, focusing on proteasome components, chaperones, deubiquitinases, and ubiquitin ligases.
For targeted validation, western blotting can confirm changes in key proteins like p62/Ref(2)P (autophagy adapter) and poly-ubiquitinated proteins . Immunohistochemistry can visualize protein aggregates in situ. RNA-seq analysis in parallel helps distinguish between transcriptional and post-transcriptional regulation. This multi-omics approach provides comprehensive insights into how Ube2d2b shapes the proteome and which protein networks are most vulnerable to its dysregulation.

How should I interpret changes in poly-ubiquitinated protein levels following Ube2d2b manipulation?

Interpreting changes in poly-ubiquitinated protein levels requires careful consideration of multiple factors. An increase in poly-ubiquitinated proteins following Ube2d2b knockdown, as observed in both soluble and insoluble fractions in Drosophila models , typically indicates impaired protein degradation rather than increased ubiquitination activity. This accumulation suggests that Ube2d2b is necessary for the proper targeting of ubiquitinated proteins to the proteasome.
When analyzing such data, distinguish between changes in different ubiquitin chain types (K48, K63, K11) using linkage-specific antibodies, as each signifies different cellular processes. Additionally, parallel assessment of proteasome activity helps determine whether increased poly-ubiquitinated proteins result from decreased Ube2d2b-mediated ubiquitination or downstream proteasomal dysfunction. Finally, assess p62/Ref(2)P levels, as their increase alongside poly-ubiquitinated proteins suggests activation of compensatory autophagy pathways in response to proteasomal insufficiency .

What proteomics approaches are most effective for identifying Ube2d2b substrates?

For identifying Ube2d2b substrates, a multi-faceted proteomics approach is most effective. Begin with quantitative proteomics comparing wild-type and Ube2d2b-deficient tissues using TMT labeling to identify proteins that accumulate upon Ube2d2b loss . Follow with ubiquitin remnant profiling (K-ε-GG) to identify specific ubiquitination sites that decrease upon Ube2d2b depletion. To distinguish direct from indirect substrates, employ in vitro ubiquitination assays with purified components followed by mass spectrometry.
Proximity labeling methods like BioID or TurboID with Ube2d2b as bait can identify proteins that physically interact with Ube2d2b in living cells. For validation, analyze candidate substrates in cell-based systems using cycloheximide chase experiments to measure protein half-life in the presence or absence of Ube2d2b. Finally, reconstitute the ubiquitination reaction in vitro with purified components to confirm direct substrate modification by Ube2d2b working with specific E3 ligases.

How do age-related changes in Ube2d2b expression correlate with proteostasis decline?

Analysis of age-related changes in Ube2d2b should evaluate protein expression across different age groups using western blotting with age-matched controls. Studies in Drosophila show that UBE2D/effete protein levels decline with age in skeletal muscle, correlating with increased insoluble poly-ubiquitinated proteins . Statistical analysis should use regression models to establish correlation strength between Ube2d2b levels and markers of proteostasis decline.
Importantly, research shows that Ube2d2b knockdown in young animals reproduces part of the proteomic changes that normally occur in aged muscles, suggesting that the age-related decrease in Ube2d2b contributes directly to reshaping the aged proteome . When interpreting these correlations, consider that some proteins upregulated by both aging and Ube2d2b deficiency are proteostasis regulators (e.g., chaperones) that may represent adaptive stress responses to loss of proteostasis rather than direct Ube2d2b substrates.

Comparative Properties of Ube2d2b Across Species

Ube2d2b-Modulated Proteins Identified Through Proteomics