Ubiquitin Transfer from the E2 Perspective: Why is UbcH5 So Promiscuous?
Open accessAbstract
Protein ubiquitination is a regulatory process that influences nearly every aspect of eukaryotic cell biology. Pathways that range from cell-cycle progression and differentiation to DNA repair to vesicle budding all rely on regulated modification of target proteins by ubiquitin. Target proteins can be tagged by a single molecule of ubiquitin or modified by ubiquitin polymers that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted. Surprisingly, little is understood regarding mechanisms of protein ubiquitination and how poly-ubiquitin chains are synthesized. Simple models to explain ubiquitin transfer have dominated the literature, but recent work suggests basic assumptions as to how proteins assemble to facilitate protein ubiquitination and poly-ubiquitin chain synthesis should be reexamined. This is particularly necessary for understanding the roles played by E2 ubiquitin-conjugating enzymes, a central protein component in all ubiquitin transfer reactions. In particular, UbcH5, a canonical E2 protein that is active in a broad number of in vitro ubiquitin transfer reactions, is capable of binding ubiquitin noncovalently on a surface distinct from its active site. This unique property allows activated UbcH5 approximately Ub complexes to self-assemble and has a profound influence on poly-ubiquitin chain synthesis.
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The UBE2D groove engagement may contribute to UBE2D's ability to ubiquitylate a broad range of substrates.
“This may contribute to UBE2D’s ability to ubiquitylate a broad range of substrates31.”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [31]
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Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler
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“Jointly, these observations could indicate more promiscuous and thereby more permissive catalytic activation of UBE2D1 by TRIM21, in agreement with earlier findings (43).”
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Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?
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Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes
Taylor, J. D., Barrett, N., Cuesta, S. M., Cassidy, K. B., Pachl, F., Dodgson, J., Patel, R. J., Eriksson, T. M., Riley, A., Burrell, M. R., et al. (2024) · Communications Biology · doi:10.1038/s42003-024-06803-4 · Cited by 19 · see citing works
“E2D1 was chosen due to its widespread expression across human cell lines, its reported promiscuity and its ability to create degradation-speci fi c (K 48 -Ub) polyubiquitin chains 23 – 25 .”
ATP-driven processes of peroxisomal matrix protein import
Schwerter, D., Grimm, I., Platta, H. W., & Erdmann, R. (2016) · Biological Chemistry · doi:10.1515/hsz-2016-0293 · Cited by 18 · see citing works
Diversity of structure and function in Cullin E3 ligases
Lin, C. P., & Komives, E. A. (2024) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2024.102879 · Cited by 17 · see citing works
Meijer, I. M. J., van Rotterdam, W., van Zoelen, E. J. J., & van Leeuwen, J. E. M. (2012) · Cellular Signalling · doi:10.1016/j.cellsig.2012.11.008 · Cited by 17 · see citing works
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“Despite the argument of UbcH5 a rather unspecific and therefore likely to light up in in vitro ubiquitination assays [22], the conjugation to TRAF6 is specific to UbcH5c, but not other members of the UbcH5 family, is highlighting the unique critical role of UbcH5c.”
BackgroundStructural aspects of multi-domain RING/Ubox E3 ligases in DNA repair
Hibbert, R. G., Mattiroli, F., & Sixma, T. K. (2009) · DNA repair · doi:10.1016/j.dnarep.2009.01.014 · Cited by 15 · see citing works
A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes
Lu, X., Malley, K. R., Brenner, C., Koroleva, O., Korolev, S., & Downes, B. P. (2016) · Nature Communications · doi:10.1038/ncomms12580 · Cited by 14 · see citing works
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“During this modification, E2 determines the processivity and topology of polyubiquitin chain formation, which ultimately regulates the fate, function, and interaction of target proteins [9,10].”
BackgroundDeng, Y., Jiang, B., Rankin, C. L., et al. (2018) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2018.08.002 · Cited by 12 · see citing works
“UbcH5a is a major E2 protein that is active in a wide-ranging number of in vitro ubiquitination reactions and binds Ub non-covalently on a surface that is distinct from its SH-containing active site [21].”
Mikami, T., Majima, S., Song, H., et al. (2023) · ACS Central Science · doi:10.1021/acscentsci.3c00389 · Cited by 11 · see citing works
Lord, S. O., Dawson, P. W. J., Chunthorng-Orn, J., Ng, J., Baehr, L. M., Hughes, D. C., Sridhar, P., Knowles, T. J., Bodine, S. C., & Lai, Y. (2024) · Biochemistry and Biophysics Reports · doi:10.1016/j.bbrep.2023.101636 · Cited by 10 · see citing works
“UBE2D families are able to generate all eight different linkage types of polyubiquitin chains [34].”
BackgroundLass, A., Cocklin, R., Scaglione, K. M., Skowyra, M. L., Korolev, S., Goebl, M. G., & Skowyra, D. (2011) · Cell Division · doi:10.1186/1747-1028-6-7 · Cited by 10 · see citing works
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UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168
Yalçin, Z., Lam, S. Y., Peuscher, M. H., van der Torre, J., Zhu, S., Iyengar, P. V., Salas‐Lloret, D., de Krijger, I., Moatti, N., van der Lugt, R., et al. (2024) · Nature Communications · doi:10.1038/s41467-024-49431-6 · Cited by 7 · see citing works
“UBE2D3 is known to have promiscuousactivityinvitro,butitsinvivorolesarelesswellde fi ned 14 , with no known roles at uncapped telomeres or in NHEJ.”
ZNFX1 uses two-component ubiquitin circuitry to quarantine viral RNA
Squair, D. R., Rivers, E., Sowar, H., Balci, A., Harmo, R., Wright, D. J., Beniwal, G., Soetens, M., Mathur, S., Tollervey, A., et al. (2026) · Molecular Cell · doi:10.1016/j.molcel.2026.02.015 · Cited by 5 · see citing works
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SPOP inhibits BRAF-dependent tumorigenesis through promoting non-degradative ubiquitination of BRAF
Feng, K. M., Shi, Q., Jiao, D., Chen, Y., Yang, W., Su, K., Wang, Y., Huang, Y., Zhang, P., Li, Y., et al. (2022) · Cell & Bioscience · doi:10.1186/s13578-022-00950-z · Cited by 6 · see citing works
“Proteins can be tagged with mono-ubiquitin or modified by polyUb chains that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted [25].”
BackgroundSingle-Turnover RING/U-Box E3-Mediated Lysine Discharge Assays
Buetow, L., Gabrielsen, M., & Huang, D. T. C. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-8706-1_2 · Cited by 6 · see citing works
“Additionally, most RING/U-box E3s react with UbcH5B~Ub and substrate lysine modification is fairly promiscuous with this E2 in vitro [16].”
BackgroundNMR characterization of foldedness for the production of E3 RING domains
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“This discrepancy can be explained by the high processivity of E2D2 in vitro [51] and potentially by a very weak and non-physiological affinity of MuRF1 for E2D2 due to the presence of the canonical Phe.”
A Modular Turn‐On Strategy to Profile E2‐Specific Ubiquitination Events in Living Cells
Hill, C. J., Datta, S., McCurtin, N. P., Kimball, H. Z., Kingsley, M. C., Bayer, A. L., Martin, A. C., Peng, Q., Weerapana, E., & Scheck, R. A. (2024) · Angewandte Chemie International Edition · doi:10.1002/anie.202319579 · Cited by 3 · see citing works
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Román-Trufero, M., Ito, C. M., Pedebos, C., Magdalou, I., Wang, Y.-F., Karimi, M. M., Moyon, B., Webster, Z., Gregorio, A. D., Azuara, V., et al. (2020) · Molecular Biology and Evolution · doi:10.1093/molbev/msaa060 · Cited by 3 · see citing works
“The promiscuous activity of UBE2D3 is an intrinsic function of the protein (Brzovic and Klevit, 2006) that is highly conserved, as evidenced by the very high homology exhibited by UBE2D3 orthologues across eukaryotic lineages.”
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The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth
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“UbcH5c was selected due to its recognized ’ promiscuous ’ activity, capable of donating active Ub to a broad spectrum of E3 ligases (31).”
Molecular characterization of the E2 conjugating enzyme LinfUbc13 in Leishmania infantum
da Silva, E. V. R., Torres, C., Ribeiro, H. N. E., de Correia, C. R. S. T. B., de Castro, T. D. O., Mancin, G. D. C., Venancio, M. G. Z., Baqui, M. M. A., Teixeira, F. R., & Gomes, M. D. (2024) · Archives of Biochemistry and Biophysics · doi:10.1016/j.abb.2024.110272 · Cited by 2 · see citing works
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A novel type of gel-like proteasome condensate induced by toxic protein aggregatesPreprint
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Biochemischer Vergleich von Proteasomen aus Hirngewebe junger und alter Ratten
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“(Brzovic and Klevit 2006; Ravid and Hochstrasser 2008; van Wijk and Timmers 2010).”
The Role of UBE2-Conjugating Enzymes in the Mechanism of MuRF1 UbiquitylationPreprint
Dawson, P. W. J., Baehr, L. M., Hughes, D. C., Knowles, T. J., Sridhar, P., Bodine, S. C., & Lai, Y. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.06.13.495268
A Modular Turn‐On Strategy to Profile E2‐Specific Ubiquitination Events in Living Cells
Hill, C. J., Datta, S., McCurtin, N. P., Kimball, H. Z., Kingsley, M. C., Bayer, A. L., Martin, A. C., Peng, Q., Weerapana, E., & Scheck, R. A. (2024) · Angewandte Chemie · doi:10.1002/ange.202319579
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E2 variants for probing E3 ubiquitin ligase activities
Du, J., Andree, G. A., Horn‐Ghetko, D., Stier, L. J., Singh, J., Kostrhon, S., Kiss, L., Mann, M., Sidhu, S. S., & Schulman, B. A. (2026) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2524899122
“UBE2D3 is even more promiscuous, with a single surface that interacts with members of all well - characterized families of E3 ligases (67 ).”
- Authors
- Brzovic, P. S., & Klevit, R. E.
- Affiliations
- University of Washington (US)
- ORCID
- Klevit, Rachel E.
- Venue
- Cell Cycle
- Publisher
- Informa UK Limited
- Published
- 2006-12-13
- Volume
- 5
- Issue
- 24
- Pages
- 2867-2873
- ISSN
- 1538-4101 (ISSN-L)
- 1551-4005
- OpenAlex
- W2064655213
- Cited by
- 89
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 9 · 2023: 3 · 2022: 4 · 2021: 3 · 2020: 4 · 2019: 4 · 2018: 4 · 2017: 3 · 2016: 3 · 2015: 3 · 2014: 5 · 2013: 3 · 2012: 7
- FWCI
- 1.90
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Cell Cycle (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute (R01CA079953)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Keywords
- ubiquitination, E2 ubiquitin-conjugating enzyme, ubiquitin transfer, ubiquitin linkage specificity
- MeSH terms
- Amino Acid Sequence, Humans, Molecular Sequence Data, Transcription Factors, Protein Structure, Secondary, BRCA1 Protein, Ubiquitin, Ubiquitin-Conjugating Enzymes
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