UBE2L3 Human His
Description
Research Applications and Experimental Findings
UBE2L3 Human His is widely used in studying ubiquitination mechanisms, cell cycle regulation, and immune responses:
Cell Cycle Regulation
-
S-Phase Control: Depletion of UBE2L3 prolongs S-phase duration, indicating its role in DNA replication and checkpoint control .
-
Mechanism: Regulates the degradation of cell cycle inhibitors (e.g., Chk1) and DNA repair proteins (e.g., 53BP1) .
Immune Modulation
-
IL-1β Regulation: UBE2L3 suppresses pro-IL-1β ubiquitination via TRIP12/AREL1, reducing inflammation .
-
NF-κB Signaling: Collaborates with LUBAC to linearly ubiquitinate NEMO, enhancing TNFα-driven NF-κB activation .
Cancer and Drug Resistance
-
AML Prognosis: High UBE2L3 expression correlates with drug resistance and poor prognosis in acute myeloid leukemia (AML) .
-
Tumor Progression: Promotes evasion of apoptosis in hepatocellular carcinoma (HCC) and cervical cancer by degrading p53 .
Disease Associations
UBE2L3 Human His is implicated in multiple diseases through its role in ubiquitination:
Interaction Partners and Pathway Involvement
UBE2L3 Human His interacts with diverse E3 ligases and signaling proteins:
Clinical and Therapeutic Implications
-
Drug Target Potential: Inhibitors of UBE2L3 (e.g., dimethyl fumarate) may suppress NF-κB-driven inflammation .
-
Biomarker Utility: Elevated UBE2L3 expression in AML patient samples predicts drug resistance and relapse .
Experimental Validation
Key Studies:
-
Cell Cycle Arrest: UBE2L3 depletion in HL60 cells extends S-phase duration, validated via flow cytometry .
-
IL-1β Suppression: Ube2l3-deficient macrophages show 2–3-fold elevated IL-1β levels post-LPS stimulation .
-
AML Drug Resistance: UBE2L3 overexpression in HL60/ADR cells correlates with Adriamycin resistance .
Product Specs
DNPPYDKGAFRIEINFPAEYPFKPPKITFKTKIYHPNIDEKGQVCLPVISAEN
WKPATKTDQVIQSLIALVNDPQPEHPLRADLAEEYSKDRKKFCKNAEEFT
KKYGEKRPVD.
Q&A
What structural features of UBE2L3 Human His make it suitable for ubiquitination assays?
UBE2L3 Human His is a 154–162 amino acid recombinant protein (18.9 kDa) with a hexahistidine tag for purification . Its catalytic core contains a conserved cysteine residue (C86) critical for thioester bond formation with ubiquitin . Unlike other E2 enzymes, UBE2L3 lacks D87 and D117 residues required for lysine reactivity, enabling unique E3 ligase partnerships with HECT/RBR-type enzymes rather than RING-domain ligases . Researchers leverage this structural specificity to study non-canonical ubiquitination pathways, particularly linear ubiquitin chain assembly in NF-κB signaling . For activity validation, perform SDS-PAGE with Coomassie staining (purity >90%) followed by in vitro ubiquitination assays using HOIP/HOIL-1L complexes .
How do researchers validate UBE2L3 activity in experimental systems?
Standard protocols involve three validation tiers:
-
Biochemical: Measure ubiquitin-thioester formation via non-reducing SDS-PAGE with E1 enzyme, ATP, and ubiquitin .
-
Functional: Co-incubate with LUBAC complex (HOIP/HOIL-1L) and NEMO substrate; detect linear ubiquitination via anti-linear Ub antibodies (e.g., MILAN-3) .
-
Cellular: Transfect UBE2L3 siRNA into HEK293/HeLa cells and quantify TNFα-induced IL8/IP-10 expression via qPCR . Activity loss upon C86A mutation confirms specificity .
What are common experimental applications of UBE2L3 Human His?
How to reconcile UBE2L3’s dual roles in promoting tumor growth vs. enhancing chemosensitivity?
Contradictory evidence exists:
-
Pro-tumor: UBE2L3 drives NSCLC proliferation via Skp2-mediated p27kip1 degradation (HR = 2.41, p = 0.007) .
-
Anti-tumor: UBE2L3 knockdown reduces Adriamycin resistance in AML (IC50 decrease 48.2% vs. control) .
Resolution strategies:
-
Context-dependent analysis: Tissue-specific E3 ligase partnerships alter outcomes. In AML, UBE2L3 collaborates with E6AP to degrade p53 , whereas in NSCLC, it partners with Skp2 .
-
Pathway modulation: CRISPR-Cas9 knockout of Skp2 in A549 cells abolishes UBE2L3’s pro-growth effect (cell cycle arrest at G1: 58.3% vs. 22.1% in controls) .
What experimental designs address UBE2L3’s role in TNFα/NF-κB signaling?
Stepwise approach:
-
Co-immunoprecipitation: Confirm UBE2L3-LUBAC interaction in HEK293T lysates .
-
Linear ubiquitination assay: Use 0.5 µM HOIP, 2 mM ATP, and 10 µg/ml ubiquitin; terminate with 50 mM DTT .
-
Functional silencing: Transfect siRNA (10 nM, 48 hr) and stimulate with 20 ng/ml TNFα; measure NF-κB nuclear translocation via EMSA .
Critical controls:
-
Catalytically inactive UBE2L3 (C86A)
-
Rescue experiments with siRNA-resistant UBE2L3 plasmid
How does UBE2L3 influence drug resistance mechanisms in leukemia?
In HL60/ADR cells:
| Parameter | UBE2L3-High | UBE2L3-Knockdown |
|---|---|---|
| Adriamycin IC50 | 12.7 µM | 6.5 µM |
| Apoptosis rate | 18.4% | 43.1% |
| p53 expression | 0.32-fold | 1.87-fold |
Mechanistically, UBE2L3 stabilizes E6AP, which ubiquitinates p53 for proteasomal degradation (K48 linkage) . To reverse resistance:
-
Inhibit UBE2L3-E6AP interaction using NSC-697923 (IC50 = 3.8 µM) .
-
Combine Adriamycin with MG132 (proteasome inhibitor) to restore p53 levels .
Resolving low ubiquitination efficiency in in vitro assays
Problem: <30% substrate ubiquitination despite optimal UBE2L3 concentrations.
Solutions:
-
Buffer optimization: Include 2 mM Mg-ATP and 1 mM DTT to stabilize E2-E3 interactions .
-
Ubiquitin priming: Pre-charge UBE2L3 with ubiquitin (30 min, 37°C) before adding E3 ligase .
-
Mutation analysis: Test C86A mutant to confirm activity dependence on catalytic cysteine .
Discrepancies in UBE2L3 expression levels across cancer types
Data conflicts:
-
Downregulation: Breast cancer (0.45-fold, TCGA data)
Investigative framework:
-
E3 ligase profiling: RNA-seq to identify co-expressed E3s (e.g., Skp2 in NSCLC vs. BRCA1 in breast cancer).
-
Substrate trapping: Use UBE2L3-C86A mutant to capture transient interactors via His-tag pulldown .
Non-canonical ubiquitination in immune signaling
Recent work implicates UBE2L3 in K63-linked ubiquitination of RIP1, enhancing TNFα-induced necroptosis (EC50 = 8.2 ng/ml TNFα) . Key experiments:
-
Necrosome isolation: RIP1/RIP3/MLKL co-IP in HT-29 cells
-
Linkage-specific Ub profiling: TUBE2 domains for K63 vs. linear chains
UBE2L3 as a biomarker for therapeutic resistance
Validation pipeline:
Product Science Overview
Ubiquitin-Conjugating Enzyme E2L 3, also known as UBE2L3, is a member of the E2 ubiquitin-conjugating enzyme family. This enzyme plays a crucial role in the ubiquitination process, which is an essential cellular mechanism for targeting abnormal or short-lived proteins for degradation. The human recombinant version of this enzyme, tagged with a His tag, is widely used in research to study its function and interactions.
UBE2L3 is characterized by its ability to accept ubiquitin from the E1 ubiquitin-activating enzyme and transfer it to target proteins in conjunction with E3 ubiquitin ligases. The enzyme specifically acts with HECT-type and RBR family E3 ubiquitin-protein ligases, but does not function with most RING-containing E3 ubiquitin-protein ligases due to its lack of intrinsic E3-independent reactivity with lysine . In vitro, UBE2L3 catalyzes ‘Lys-11’-linked polyubiquitination, which is involved in the selective degradation of short-lived and abnormal proteins .
The modification of proteins with ubiquitin is a critical cellular process that regulates various aspects of cell biology, including protein degradation, cell cycle progression, and DNA repair. UBE2L3 has been demonstrated to participate in the ubiquitination of key regulatory proteins such as p53, c-Fos, and the NF-kB precursor p105 . This highlights its importance in maintaining cellular homeostasis and responding to cellular stress.
The human recombinant UBE2L3, tagged with a His tag, is produced in Escherichia coli and purified to a high degree of purity. The His tag facilitates the purification process and allows for easy detection and quantification of the protein in various assays. This recombinant protein is used in research to study the enzyme’s function, interactions, and role in ubiquitination pathways .
Recombinant UBE2L3 is utilized in various experimental setups, including in vitro ubiquitination assays, protein-protein interaction studies, and structural analysis. Its role in the ubiquitination of specific substrates makes it a valuable tool for understanding the molecular mechanisms underlying protein degradation and regulation. Additionally, studying UBE2L3 can provide insights into the development of therapeutic strategies for diseases associated with dysregulated ubiquitination, such as cancer and neurodegenerative disorders .
