ArticleThe EMBO journal2012
Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria.
Article in The EMBO journal, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed, 56 citations in OpenAlex.
- A pan-cancer analysis of MARCH8: molecular characteristics, clinical relevance, and immuno-oncology features.Cancer biology & therapy · 2025Article
- SNARE-Mediated Membrane Fusion Probed Using a Synthetic Organelle in the Living Bacterium.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Genetic Code Expansion Approaches to Decipher the Ubiquitin Code.Chemical reviews · 2024Review
- Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions.International journal of molecular sciences · 2024Article
- A Bacterial Platform for Studying Ubiquitination Cascades Anchored by SCF-Type E3 Ubiquitin Ligases.Biomolecules · 2024Article
- CRL4Life science alliance · 2024Article
- Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.The EMBO journal · 2024Article
- Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.ACS central science · 2022Article
- Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class.Chembiochem : a European journal of chemical biology · 2021Review
- Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations.Human molecular genetics · 2021Article
- When MARCH family proteins meet viral infections.Virology journal · 2021Review
- Mono-ubiquitination of Rabphilin 3A by UBE3A serves a non-degradative function.Scientific reports · 2021Article
- A novel UBE3A sequence variant identified in eight related individuals with neurodevelopmental delay, results in a phenotype which does not match the clinical criteria of Angelman syndrome.Molecular genetics & genomic medicine · 2020Article
- An Integrative Synthetic Biology Approach to Interrogating Cellular Ubiquitin and Ufm Signaling.International journal of molecular sciences · 2020Article
- ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection.Nature microbiology · 2019Article
- How the 26S Proteasome Degrades Ubiquitinated Proteins in the Cell.Biomolecules · 2019Review
- Remodeling Membrane Binding by Mono-Ubiquitylation.Biomolecules · 2019Article
- A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins.Nature communications · 2019Article
- Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase.Nature chemical biology · 2019Article
- Excessive UBE3A dosage impairs retinoic acid signaling and synaptic plasticity in autism spectrum disorders.Cell research · 2018Article
Corrections and comments
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Authors and funding
14 authors at 5 institutions in 3 countries.
Funding
Abstract
Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.