UBA3 Human
Description
UBA3 is fused to a 24 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
Product Specs
Q&A
What is the primary biochemical function of UBA3 in the NEDD8 activation pathway?
UBA3 partners with APPBP1 to form the NAE heterodimer, which catalyzes ATP-dependent activation of NEDD8. This process involves three steps:
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Adenylation: UBA3 binds ATP and NEDD8, forming a NEDD8-AMP intermediate.
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Thioester bond formation: NEDD8 is transferred to UBA3’s catalytic cysteine residue.
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E2 enzyme charging: NEDD8 is conjugated to the E2 enzyme UBE2M (Ubc12) .
Methodological Insight: To confirm UBA3’s role, researchers often use siRNA knockdown or CRISPR-Cas9 knockout models. For example, silencing UBA3 in leukemia cells (K562, U937) disrupts cullin neddylation, leading to cell cycle arrest and apoptosis . Western blotting with antibodies targeting neddylated cullins (e.g., CUL1, CUL3) validates functional loss .
How does the UBA3-APPBP1 heterodimer compare to other E1 enzymes structurally?
UBA3 belongs to the E1 enzyme family but uniquely requires heterodimerization with APPBP1 for activity. Structural studies reveal:
| Feature | UBA3-APPBP1 | Other E1s (e.g., UBA1) |
|---|---|---|
| Subunit Composition | Heterodimer | Monomeric or homodimeric |
| Catalytic Core | UBA3 alone suffices | Single subunit |
| Scaffold Role | APPBP1 enhances kinetics | Not applicable |
| E2 Binding Domain | β-grasp fold (UBA3) | Ubiquitin-like domain |
Methodological Insight: Quantitative FRET assays and AESOP computational modeling have been pivotal in dissecting subunit contributions. For instance, truncating APPBP1 reduces NEDD8 activation rates by 60%, confirming its non-catalytic scaffolding role .
What cellular processes are most sensitive to UBA3 inhibition?
UBA3-mediated neddylation regulates:
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Cell cycle progression: Neddylation activates cullin-RING ligases (CRLs), which degrade cell cycle inhibitors (e.g., p21, p27) .
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DNA damage repair: CRLs control substrates like CDT1, preventing re-replication .
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Embryogenesis: UBA3 knockout models exhibit embryonic lethality due to defective mitotic signaling .
Methodological Insight: To assess pathway sensitivity, dose-response assays with NAE inhibitors (e.g., MLN4924) are performed. IC50 values for cell viability (e.g., 10–50 nM in leukemia cells) correlate with CRL substrate accumulation .
How do UBA3 mutations confer resistance to NAE inhibitors like MLN4924?
Resistance mutations (e.g., I310N, Y352H) arise under prolonged MLN4924 exposure. Biochemical analyses reveal:
| Mutation | ATP Affinity (Km) | NEDD8 Affinity (Km) | MLN4924 IC50 Shift |
|---|---|---|---|
| Wild-type | 15 µM | 0.8 µM | 1× (baseline) |
| I310N | 6 µM (-60%) | 2.5 µM (+212%) | 12× |
| Y352H | 9 µM (-40%) | 3.1 µM (+288%) | 8× |
Mechanism: Mutations increase ATP binding efficiency while reducing NEDD8 affinity, allowing residual neddylation under inhibitor pressure .
Methodological Insight: To identify resistance alleles, perform long-term MLN4924 selection (6+ months) on leukemia cells, followed by whole-exome sequencing. Validate using in vitro ATP/NEDD8 competition assays .
What experimental strategies elucidate UBA3’s interaction with E2 enzymes?
UBA3’s β-grasp domain recruits UBE2M via electrostatic interactions. Key approaches include:
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Site-directed mutagenesis: Alanine substitution of UBA3 residues (e.g., R228, K231) disrupts UBE2M binding .
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Isothermal titration calorimetry (ITC): Measures binding affinity (Kd = 120 nM for wild-type UBA3-UBE2M) .
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Cryo-EM: Resolves conformational changes during NEDD8 transfer .
Example Workflow:
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Clone UBA3 and UBE2M into bacterial expression vectors.
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Purify proteins via affinity chromatography.
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Conduct ITC at 25°C in 20 mM Tris (pH 7.5), 150 mM NaCl.
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Analyze data using MicroCal PEAQ-ITC software.
How can researchers reconcile contradictory data on UBA3’s role in non-canonical pathways?
Some studies suggest UBA3 neddylates non-cullin targets (e.g., EGFR, STAT3), but others attribute these findings to off-target effects. Resolution strategies:
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Conditional knockout models: Use Cre-lox systems to delete UBA3 in specific cell types, minimizing compensatory mechanisms.
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Chemical biology: Employ NEDD8 variants (e.g., NEDD8-Dha) for covalent trapping, identifying direct substrates via mass spectrometry .
Data Interpretation: Cross-validate findings with multiple E1 inhibitors (e.g., MLN4924 vs. pan-E1 blockers) to distinguish NAE-specific effects .
What computational tools predict UBA3’s structural dynamics during catalysis?
The AESOP framework integrates:
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Electrostatic potential maps: Predict UBA3-APPBP1 interface stability.
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Molecular dynamics (MD) simulations: Model ATP/NEDD8 binding kinetics .
Workflow:
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Generate homology models using SWISS-MODEL.
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Simulate ATP binding with GROMACS (50 ns trajectories).
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Validate with hydrogen-deuterium exchange mass spectrometry (HDX-MS).
How to address low yield in recombinant UBA3-APPBP1 expression?
Problem: The UBA3-APPBP1 heterodimer often misfolds in E. coli.
Solutions:
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Use bicistronic vectors (e.g., pET-Duet) for co-expression.
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Optimize induction at 18°C with 0.2 mM IPTG.
What controls are critical when profiling UBA3 activity in clinical samples?
Recommendations:
Product Science Overview
Ubiquitin-Like Modifier Activating Enzyme 3 (UBA3) is a crucial component in the ubiquitin-proteasome system, which is responsible for the degradation of proteins within the cell. This enzyme is part of the E1 ubiquitin-activating enzyme family and plays a significant role in the neddylation pathway, which is essential for various cellular processes, including cell division, signaling, and embryogenesis .
UBA3 is encoded by the UBA3 gene located on chromosome 3p14.1 . The gene produces a protein that consists of 442 amino acids and shares 43% sequence identity with its yeast homolog . The UBA3 protein forms a heterodimer with APPBP1 (amyloid beta precursor protein binding protein 1), which is necessary for its function .
UBA3, in conjunction with APPBP1, forms the NEDD8-activating enzyme (NAE). This enzyme complex is responsible for the activation of NEDD8, a ubiquitin-like protein. The activation process involves the adenylation of the C-terminal glycine residue of NEDD8 with ATP, followed by the formation of a thioester bond between NEDD8 and the catalytic cysteine residue of UBA3 . This activated NEDD8 is then transferred to specific target proteins, regulating their stability and function.
The neddylation pathway, mediated by UBA3, is crucial for the regulation of the cullin-RING ubiquitin ligases (CRLs). These ligases are involved in the ubiquitination and subsequent degradation of various proteins, thereby controlling numerous cellular processes . Dysregulation of this pathway can lead to various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions.
Research on UBA3 has provided insights into its role in cellular homeostasis and disease. The recombinant form of UBA3 is used in various studies to understand its function and to develop potential therapeutic interventions. For instance, inhibitors targeting the NEDD8-activating enzyme are being explored as potential treatments for cancer, as they can disrupt the degradation of proteins that promote tumor growth .
