GO:0061631 ubiquitin conjugating enzyme activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0061631 (ubiquitin conjugating enzyme activity) describes the isopeptide transfer of ubiquitin from an E2-ubiquitin thioester to a substrate lysine, a central step in ubiquitin-proteasome signaling.
• E2 enzymes (UBE2 family) form a thioester bond between their catalytic cysteine and the C-terminal glycine of ubiquitin, then cooperate with E3 ligases to attach ubiquitin to targets.
• Dysregulated E2 activity is linked to acute myeloid leukemia, renal fibrosis, endothelial signaling defects, and multiple solid tumors.
• UBE2N, UBE2T, UBE2C, UBE2Q2, and UBE2V1 are among the most studied E2 enzymes in human disease and cell biology.
• Plant PEX4 is a ubiquitin-conjugating enzyme required for peroxisomal protein transport, showing the pathway is conserved beyond humans.
• CRISPR knockout, point-mutation, knock-in, and overexpression models are essential to test whether a specific E2 gene causally drives a phenotype.
Description
Ubiquitin conjugating enzyme activity (GO:0061631) is the molecular function that transfers ubiquitin from an E2-ubiquitin thioester to a target protein, forming a new ubiquitin-substrate linkage. This reaction is the second enzymatic step of the ubiquitin-proteasome system, following E1-mediated ubiquitin activation and preceding E3-mediated substrate selection. Because E2 enzymes determine which ubiquitin chain linkages are built and which E3 ligases can be engaged, they sit at the decision point between protein stability, localization, and signaling. Researchers study GO:0061631 to understand how cells control proteostasis, DNA repair, immune signaling, and cell-cycle progression. In cancer, E2 enzymes such as UBE2T and UBE2C are frequently overexpressed and correlate with poor prognosis, making them attractive therapeutic targets. In non-cancer contexts, UBE2Q2 protects renal tubules from fibrosis, and UBE2V1 enables endothelial responses to fibroblast growth factor signaling. Plant PEX4 demonstrates that ubiquitin-conjugating activity is also required for peroxisomal protein import, underscoring the deep evolutionary conservation of this function.
ubiquitin conjugating enzyme activity At A Glance
| GO ID | GO:0061631 |
|---|---|
| GO term | ubiquitin conjugating enzyme activity |
| Ontology | molecular_function |
| Synonym | E2, HECT E3 |
| Major function | Transfer of ubiquitin from an E2-ubiquitin thioester to a substrate protein |
| Catalytic residue | Cysteine sulfhydryl group forming a thioester with ubiquitin C-terminal glycine |
| Pathway context | Second step of the ubiquitin-proteasome system, after E1 activation and before E3-mediated substrate selection |
| Representative enzymes | UBE2N, UBE2T, UBE2C, UBE2Q2, UBE2V1, PEX4 |
What Is GO:0061631?
GO:0061631 describes the isenergetic transfer of ubiquitin from one protein to another, where both the donor (X-ubiquitin) and acceptor (Y-ubiquitin) linkages are thioester bonds between the C-terminal glycine of ubiquitin and a cysteine sulfhydryl group. In practice, an E2 enzyme charged with ubiquitin via a thioester bond interacts with an E3 ligase to transfer ubiquitin to a substrate lysine, forming an isopeptide bond. The term captures the catalytic activity of E2 enzymes, not the upstream E1 activation or downstream substrate recognition steps.
Why Is ubiquitin conjugating enzyme activity Important in Cell Biology?
Ubiquitin conjugating enzyme activity is essential because it determines the efficiency and linkage specificity of ubiquitin transfer, thereby controlling protein half-life, localization, and interaction networks. Disruption of E2 function contributes to leukemia, fibrosis, endothelial dysfunction, and tumor progression, while plant PEX4 loss impairs peroxisomal transport. Because E2 enzymes are tractable drug targets and their activity can be measured biochemically, they are central to both mechanistic biology and translational research.
• Controls proteostasis by tagging proteins for proteasomal degradation.
• Determines ubiquitin chain linkage type and downstream signaling outcomes.
• Modulates immunoproteasome function in acute myeloid leukemia.
• Drives tumorigenesis when UBE2T is overexpressed.
• Protects renal tubulointerstitial tissue via UBE2Q2-HUWE1 cooperation.
• Enables endothelial fibroblast growth factor signaling through UBE2V1.
• Serves as a cancer biomarker, e.g., UBE2C.
• Required for peroxisomal protein transport in plants via PEX4.
• Provides a druggable node in the ubiquitin-proteasome system.
• Offers a functional readout for CRISPR-based gene editing studies.
What Happens During ubiquitin conjugating enzyme activity?
E2 charging by E1
In simple terms: An E1 enzyme hands ubiquitin to an E2 enzyme.
The E2 enzyme receives ubiquitin from an E1 via a thioester bond between its catalytic cysteine and the C-terminal glycine of ubiquitin, forming the E2-ubiquitin intermediate required for GO:0061631. This step is shared across E2 family members and is a prerequisite for substrate modification.
E3-mediated substrate recognition
In simple terms: An E3 ligase brings the target protein to the E2.
E3 ligases bind both the E2-ubiquitin thioester and the substrate, positioning the substrate lysine for nucleophilic attack. Structural diversity among E3 ligases determines which E2 enzymes and substrates are paired, thereby shaping the specificity of ubiquitin conjugating enzyme activity.
Isopeptide bond formation
In simple terms: Ubiquitin is glued onto the target protein.
The E2 catalytic cysteine transfers ubiquitin to a substrate lysine, forming an isopeptide bond and completing the reaction described by GO:0061631. Repeated cycles build polyubiquitin chains whose linkage type dictates whether the substrate is degraded or redirected.
Linkage-specific chain assembly
In simple terms: Different E2s build different ubiquitin chains.
E2 enzymes such as UBE2N and UBE2V1 cooperate to generate specific chain linkages that control immune signaling and endothelial responses. The choice of E2 therefore influences whether a substrate is targeted to the proteasome or used as a signaling scaffold.
Substrate fate and downstream effects
In simple terms: The ubiquitin tag changes what happens to the protein.
Ubiquitination can alter protein stability, localization, or interactions, and dysregulation of this step is linked to leukemia, fibrosis, and tumorigenesis. Measuring E2 activity therefore provides a functional window into disease mechanisms.
Key Genes Involved in GO:0061631 ubiquitin conjugating enzyme activity
The following genes encode E2 enzymes or directly associated factors whose catalytic or regulatory roles are supported by published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| UBE2N | E2 enzyme modulating proteostasis | Linked to immunoproteasome-positive acute myeloid leukemia |
| UBE2T | E2 enzyme in tumorigenesis | Overexpressed in multiple cancers; review highlights oncogenic roles |
| UBE2C | E2 enzyme in cell-cycle control | Potential cancer biomarker |
| UBE2Q2 | E2 enzyme cooperating with HUWE1 | Protects against renal tubulointerstitial fibrosis |
| UBE2V1 | E2 variant enabling signaling | Required for endothelial fibroblast growth factor responses |
| PEX4 | Plant E2 enzyme for peroxisomal transport | Required for efficient protein transport to peroxisomes |
| UBE2D1 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2D2 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2D3 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2E1 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2E2 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2E3 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2G1 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2G2 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2H | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
| UBE2I | E2 enzyme for SUMOylation-related transfer | General E2 function in ubiquitin-like conjugation |
| UBE2J1 | E2 enzyme for ubiquitin transfer | General E2 function in ubiquitin-proteasome system |
How Is ubiquitin conjugating enzyme activity Regulated?
Ubiquitin conjugating enzyme activity is regulated at multiple levels, including E1-mediated charging, E3 ligase pairing, and post-translational modification of E2 enzymes. Structural diversity among E3 ligases determines which E2 enzymes are recruited to specific substrates, thereby controlling the timing and location of ubiquitin transfer. In endothelial cells, UBE2V1 enables cellular responses to fibroblast growth factor signaling, illustrating how extracellular cues can influence E2-dependent processes. In acute myeloid leukemia, UBE2N modulates proteostasis in immunoproteasome-positive cells, suggesting that E2 activity is tuned to the proteotoxic state of the cell. Plant PEX4 activity is required for peroxisomal protein transport, showing that E2 regulation is integrated with organellar biogenesis.
ubiquitin conjugating enzyme activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| UBE2N | Acute myeloid leukemia | Knockout in AML cell lines; proteostasis assays |
| UBE2T | Tumorigenesis | Overexpression and knockout in cancer cell lines |
| UBE2C | Cancer biomarker | Point mutation and overexpression models |
| UBE2Q2 | Renal tubulointerstitial fibrosis | Knockout in renal tubular cells; fibrosis models |
| UBE2V1 | Endothelial FGF signaling | Knockout in endothelial cells; signaling assays |
Ubiquitin conjugating enzymes in cancer
UBE2T is overexpressed in multiple tumors and its oncogenic roles have been comprehensively reviewed, supporting its potential as a therapeutic target. UBE2C has been proposed as a cancer biomarker, with expression changes correlating with tumor progression. In acute myeloid leukemia, UBE2N modulates proteostasis in immunoproteasome-positive cells, linking E2 activity to leukemia cell survival.
E2 enzymes in fibrosis and tissue protection
UBE2Q2 participates in HUWE1-mediated protection against renal tubulointerstitial fibrosis, indicating that E2 activity can be protective in chronic kidney injury. This suggests that modulating E2 function may have therapeutic potential beyond oncology.
Endothelial signaling and vascular biology
UBE2V1 enables cellular responses toward fibroblast growth factor signaling in endothelium, connecting ubiquitin conjugating enzyme activity to vascular growth and repair. Dysregulation of this axis could contribute to angiogenesis-related pathologies.
Conserved roles in plant peroxisomes
PEX4 ubiquitin-conjugating activity is required for efficient protein transport to peroxisomes in Arabidopsis thaliana, demonstrating that GO:0061631 is essential for organelle function across kingdoms. This conservation highlights the fundamental importance of E2 enzymes in cellular logistics.
From ubiquitin conjugating enzyme activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is UBE2N required for leukemia cell survival? | CRISPR knockout in AML cell lines |
| Does UBE2T overexpression drive tumor growth? | Knock-in or overexpression in cancer cells |
| Can a point mutation in UBE2C alter its catalytic activity? | CRISPR point mutation at catalytic cysteine |
| Does UBE2Q2 protect against renal fibrosis? | Knockout mouse or renal tubular cell model |
| Is UBE2V1 needed for endothelial FGF signaling? | Knockout endothelial cells |
| Is PEX4 activity required for peroxisomal transport? | Plant knockout or point mutation |
How to Study the ubiquitin conjugating enzyme activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro ubiquitin transfer assay | Thioester and isopeptide bond formation | Measure E2 catalytic activity |
| Ubiquitin remnant proteomics | Substrate lysine ubiquitination | Identify E2 targets |
| CRISPR knockout screen | Gene requirement for phenotype | Discover essential E2 enzymes |
| Western blot with ubiquitin antibodies | Global ubiquitin conjugates | Assess pathway activity |
| Fluorescence microscopy | E2 and substrate localization | Study organelle-specific transfer |
| Co-immunoprecipitation | E2-E3 and E2-substrate interactions | Map interaction networks |
| Site-directed mutagenesis | Catalytic cysteine requirement | Validate GO:0061631 mechanism |
| RNA-seq | Transcriptional changes after E2 perturbation | Identify downstream pathways |
Biochemical ubiquitin transfer assays
In vitro assays using recombinant E1, E2, E3, ubiquitin, and substrate measure thioester formation and isopeptide bond creation, directly reporting GO:0061631 activity. These assays can be coupled with mutagenesis of the catalytic cysteine to confirm specificity.
Proteomics and ubiquitin remnant profiling
Mass spectrometry-based ubiquitin remnant profiling identifies substrate lysines modified by E2 enzymes, revealing downstream effects of altered ubiquitin conjugating enzyme activity. This approach is useful for comparing knockout versus wild-type cells.
CRISPR screening and functional genomics
Pooled CRISPR knockout screens targeting E2 genes can identify which ubiquitin conjugating enzymes are required for a phenotype, such as cancer cell proliferation or drug response. Hits can be validated with individual knockouts and rescue experiments.
Imaging and localization studies
Fluorescent tagging of E2 enzymes and substrates allows visualization of ubiquitin transfer at specific cellular sites, including peroxisomes and endothelial signaling compartments. Live-cell imaging can capture dynamic changes in E2 recruitment.
How CRISPR Can Be Used to Study GO:0061631 ubiquitin conjugating enzyme activity
Knockout
CRISPR knockout of E2 genes such as UBE2N or UBE2T can reveal whether ubiquitin conjugating enzyme activity is required for cell survival, proliferation, or stress responses. Knockout models are also used to validate hits from pooled screens.
Point Mutation
Introducing point mutations at the catalytic cysteine of an E2 enzyme abolishes thioester formation, providing a clean way to separate catalytic activity from scaffolding functions. Such mutants are valuable for testing whether a phenotype depends specifically on GO:0061631.
Knock-in
Knock-in of tagged or mutant E2 alleles allows endogenous-level expression of reporters or disease-associated variants, enabling precise studies of ubiquitin conjugating enzyme activity in a physiological context. This approach is useful for tracking E2 localization and dynamics.
Overexpression
Overexpression of E2 enzymes such as UBE2T or UBE2C can model tumor-associated gain of function and test whether increased ubiquitin conjugating enzyme activity drives oncogenic phenotypes. Overexpression models are also used to study endothelial signaling via UBE2V1.
How EDITGENE Supports ubiquitin conjugating enzyme activity Research
Researchers studying ubiquitin conjugating enzyme activity-related genes often need to determine whether a candidate gene is causally involved in a phenotype, which requires precise genetic models that isolate catalytic function from scaffolding roles. EDITGENE provides end-to-end CRISPR services to generate such models efficiently.
Contact EDITGENE today to design your custom CRISPR model for ubiquitin conjugating enzyme activity research.
Frequently Asked Questions About ubiquitin conjugating enzyme activity
What is ubiquitin conjugating enzyme activity?
It is the molecular function GO:0061631, in which an E2 enzyme transfers ubiquitin from a thioester intermediate to a substrate protein.
What genes are involved in ubiquitin conjugating enzyme activity?
Key genes include UBE2N, UBE2T, UBE2C, UBE2Q2, UBE2V1, and PEX4, among many other UBE2 family members.
What is the GO ID for ubiquitin conjugating enzyme activity?
The GO ID is GO:0061631.
How does ubiquitin conjugating enzyme activity work?
An E1 charges an E2 with ubiquitin, and an E3 ligase positions the substrate so the E2 can form an isopeptide bond.
Which diseases are linked to ubiquitin conjugating enzymes?
They are linked to acute myeloid leukemia, renal fibrosis, endothelial signaling defects, and multiple cancers.
What is the role of UBE2T in cancer?
UBE2T is overexpressed in tumors and has oncogenic roles, making it a potential therapeutic target.
How can I study ubiquitin conjugating enzyme activity in the lab?
In vitro ubiquitin transfer assays, proteomics, and CRISPR knockout models are commonly used.
What CRISPR models are available for E2 genes?
Knockout, point mutation, knock-in, and overexpression models can be generated for E2 genes.
Is ubiquitin conjugating enzyme activity conserved in plants?
Yes, PEX4 is a plant E2 enzyme required for peroxisomal protein transport.
What is the synonym for GO:0061631?
The synonyms include E2 and HECT E3.
Conclusion
Ubiquitin conjugating enzyme activity (GO:0061631) is a central molecular function that governs protein fate through the ubiquitin-proteasome system. Its dysregulation is implicated in leukemia, fibrosis, endothelial dysfunction, and cancer, making E2 enzymes important research and therapeutic targets. CRISPR-based models that knockout, mutate, knock in, or overexpress E2 genes provide the causal evidence needed to translate these observations into new treatments.
References
- 1. Ishikawa C et al.. 2025. Ubiquitin-conjugating enzyme UBE2N modulates proteostasis in immunoproteasome-positive acute myeloid leukemia.. J Clin Invest 135(10) PMID: 40371639
- 2. Toma-Fukai S et al.. 2021. Structural Diversity of Ubiquitin E3 Ligase.. Molecules 26(21) PMID: 34771091
- 3. Gao C et al.. 2024. Unraveling the Role of Ubiquitin-Conjugating Enzyme UBE2T in Tumorigenesis: A Comprehensive Review.. Cells 14(1) PMID: 39791716
- 4. Wang Z et al.. 2022. [Ubiquitin-conjugating enzyme UBE2Q2 participates in HUWE1-mediated protection on renal tubulointerstitial fibrosis].. Sheng Li Xue Bao 74(1):117-124 PMID: 35199132
- 5. Liu W et al.. 2020. The Ubiquitin Conjugating Enzyme: An Important Ubiquitin Transfer Platform in Ubiquitin-Proteasome System.. Int J Mol Sci 21(8) PMID: 32326224
- 6. Mano S et al.. 2022. Ubiquitin-conjugating activity by PEX4 is required for efficient protein transport to peroxisomes in Arabidopsis thaliana.. J Biol Chem 298(6):102038 PMID: 35595097
- 7. Xie C et al.. 2014. Ubiquitin-conjugating enzyme E2C: a potential cancer biomarker.. Int J Biochem Cell Biol 47:113-7 PMID: 24361302
- 8. Elangovan M et al.. 2022. Ubiquitin-conjugating enzyme V variant 1 enables cellular responses toward fibroblast growth factor signaling in endothelium.. FASEB J 36(1):e22103 PMID: 34921695
