Home > Institute Collections > W500 > Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B. > print

001     306244
005     20251118114556.0
024 7 _ |a 10.1016/j.jmb.2021.167276
|2 doi
024 7 _ |a pmid:34599943
|2 pmid
024 7 _ |a 0022-2836
|2 ISSN
024 7 _ |a 1089-8638
|2 ISSN
037 _ _ |a DKFZ-2025-02473
041 _ _ |a English
082 _ _ |a 610
100 1 _ |a Levin-Kravets, Olga
|b 0
245 _ _ |a Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.
260 _ _ |a Amsterdam [u.a.]
|c 2021
|b Elsevier
336 7 _ |a article
|2 DRIVER
336 7 _ |a Output Types/Journal article
|2 DataCite
336 7 _ |a Journal Article
|b journal
|m journal
|0 PUB:(DE-HGF)16
|s 1763462731_1939188
|2 PUB:(DE-HGF)
336 7 _ |a ARTICLE
|2 BibTeX
336 7 _ |a JOURNAL_ARTICLE
|2 ORCID
336 7 _ |a Journal Article
|0 0
|2 EndNote
500 _ _ |a #DKFZ-MOST-Ca196#
520 _ _ |a Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.
588 _ _ |a Dataset connected to CrossRef, PubMed, , Journals: inrepo02.dkfz.de
650 _ 7 |a Kaufman oculocerebrofacial syndrome
|2 Other
650 _ 7 |a protein-protein interaction assay
|2 Other
650 _ 7 |a ubiquitylation
|2 Other
650 _ 7 |a Chloramphenicol O-Acetyltransferase
|0 EC 2.3.1.28
|2 NLM Chemicals
650 _ 7 |a Ubiquitin-Protein Ligases
|0 EC 2.3.2.27
|2 NLM Chemicals
650 _ 2 |a Biological Assay: methods
|2 MeSH
650 _ 2 |a Chloramphenicol O-Acetyltransferase: genetics
|2 MeSH
650 _ 2 |a Chloramphenicol O-Acetyltransferase: metabolism
|2 MeSH
650 _ 2 |a Enzyme Activation
|2 MeSH
650 _ 2 |a Escherichia coli: genetics
|2 MeSH
650 _ 2 |a Escherichia coli: metabolism
|2 MeSH
650 _ 2 |a Gene Expression
|2 MeSH
650 _ 2 |a Genes, Reporter
|2 MeSH
650 _ 2 |a Protein Processing, Post-Translational
|2 MeSH
650 _ 2 |a Proteolysis
|2 MeSH
650 _ 2 |a Ubiquitin-Protein Ligases: metabolism
|2 MeSH
650 _ 2 |a Ubiquitination
|2 MeSH
700 1 _ |a Kordonsky, Alina
|b 1
700 1 _ |a Shusterman, Anna
|b 2
700 1 _ |a Biswas, Sagnik
|b 3
700 1 _ |a Persaud, Avinash
|b 4
700 1 _ |a Elias, Sivan
|b 5
700 1 _ |a Langut, Yael
|b 6
700 1 _ |a Florentin, Amir
|b 7
700 1 _ |a Simpson-Lavy, Kobi J
|b 8
700 1 _ |a Yariv, Elon
|b 9
700 1 _ |a Avishid, Reut
|b 10
700 1 _ |a Sror, Mor
|b 11
700 1 _ |a Almog, Ofir
|b 12
700 1 _ |a Marshanski, Tal
|b 13
700 1 _ |a Kadosh, Shira
|b 14
700 1 _ |a Ben David, Nicole
|b 15
700 1 _ |a Manori, Bar
|b 16
700 1 _ |a Fischer, Zohar
|b 17
700 1 _ |a Lilly, Jeremiah
|b 18
700 1 _ |a Borisova, Ekaterina
|b 19
700 1 _ |a Ambrozkiewicz, Mateusz C
|b 20
700 1 _ |a Tarabykin, Victor
|b 21
700 1 _ |a Kupiec, Martin
|b 22
700 1 _ |a Thaker, Maulik
|b 23
700 1 _ |a Rotin, Daniela
|b 24
700 1 _ |a Prag, Gali
|b 25
773 _ _ |a 10.1016/j.jmb.2021.167276
|g Vol. 433, no. 23, p. 167276 -
|0 PERI:(DE-600)1355192-9
|n 23
|p 167276
|t Journal of molecular biology
|v 433
|y 2021
|x 0022-2836
915 _ _ |a Nationallizenz
|0 StatID:(DE-HGF)0420
|2 StatID
|d 2024-12-13
|w ger
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0200
|2 StatID
|b SCOPUS
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0300
|2 StatID
|b Medline
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0199
|2 StatID
|b Clarivate Analytics Master Journal List
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)1050
|2 StatID
|b BIOSIS Previews
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0160
|2 StatID
|b Essential Science Indicators
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)1030
|2 StatID
|b Current Contents - Life Sciences
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)1190
|2 StatID
|b Biological Abstracts
|d 2024-12-13
915 _ _ |a WoS
|0 StatID:(DE-HGF)0113
|2 StatID
|b Science Citation Index Expanded
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0150
|2 StatID
|b Web of Science Core Collection
|d 2024-12-13
915 _ _ |a JCR
|0 StatID:(DE-HGF)0100
|2 StatID
|b J MOL BIOL : 2022
|d 2024-12-13
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0600
|2 StatID
|b Ebsco Academic Search
|d 2024-12-13
915 _ _ |a Peer Review
|0 StatID:(DE-HGF)0030
|2 StatID
|b ASC
|d 2024-12-13
915 _ _ |a IF >= 5
|0 StatID:(DE-HGF)9905
|2 StatID
|b J MOL BIOL : 2022
|d 2024-12-13
980 _ _ |a journal
980 _ _ |a I:(DE-He78)W500-20160331
980 _ _ |a I:(DE-He78)W510-20160331
980 1 _ |a EXTERN4COORD

LibraryCollectionCLSMajorCLSMinorLanguageAuthor
Marc 21