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000306244 0247_ $$2doi$$a10.1016/j.jmb.2021.167276
000306244 0247_ $$2pmid$$apmid:34599943
000306244 0247_ $$2ISSN$$a0022-2836
000306244 0247_ $$2ISSN$$a1089-8638
000306244 037__ $$aDKFZ-2025-02473
000306244 041__ $$aEnglish
000306244 082__ $$a610
000306244 1001_ $$aLevin-Kravets, Olga$$b0
000306244 245__ $$aSplit Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.
000306244 260__ $$aAmsterdam [u.a.]$$bElsevier$$c2021
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000306244 520__ $$aSplit 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.
000306244 588__ $$aDataset connected to CrossRef, PubMed, , Journals: inrepo02.dkfz.de
000306244 650_7 $$2Other$$aKaufman oculocerebrofacial syndrome
000306244 650_7 $$2Other$$aprotein-protein interaction assay
000306244 650_7 $$2Other$$aubiquitylation
000306244 650_7 $$0EC 2.3.1.28$$2NLM Chemicals$$aChloramphenicol O-Acetyltransferase
000306244 650_7 $$0EC 2.3.2.27$$2NLM Chemicals$$aUbiquitin-Protein Ligases
000306244 650_2 $$2MeSH$$aBiological Assay: methods
000306244 650_2 $$2MeSH$$aChloramphenicol O-Acetyltransferase: genetics
000306244 650_2 $$2MeSH$$aChloramphenicol O-Acetyltransferase: metabolism
000306244 650_2 $$2MeSH$$aEnzyme Activation
000306244 650_2 $$2MeSH$$aEscherichia coli: genetics
000306244 650_2 $$2MeSH$$aEscherichia coli: metabolism
000306244 650_2 $$2MeSH$$aGene Expression
000306244 650_2 $$2MeSH$$aGenes, Reporter
000306244 650_2 $$2MeSH$$aProtein Processing, Post-Translational
000306244 650_2 $$2MeSH$$aProteolysis
000306244 650_2 $$2MeSH$$aUbiquitin-Protein Ligases: metabolism
000306244 650_2 $$2MeSH$$aUbiquitination
000306244 7001_ $$aKordonsky, Alina$$b1
000306244 7001_ $$aShusterman, Anna$$b2
000306244 7001_ $$aBiswas, Sagnik$$b3
000306244 7001_ $$aPersaud, Avinash$$b4
000306244 7001_ $$aElias, Sivan$$b5
000306244 7001_ $$aLangut, Yael$$b6
000306244 7001_ $$aFlorentin, Amir$$b7
000306244 7001_ $$aSimpson-Lavy, Kobi J$$b8
000306244 7001_ $$aYariv, Elon$$b9
000306244 7001_ $$aAvishid, Reut$$b10
000306244 7001_ $$aSror, Mor$$b11
000306244 7001_ $$aAlmog, Ofir$$b12
000306244 7001_ $$aMarshanski, Tal$$b13
000306244 7001_ $$aKadosh, Shira$$b14
000306244 7001_ $$aBen David, Nicole$$b15
000306244 7001_ $$aManori, Bar$$b16
000306244 7001_ $$aFischer, Zohar$$b17
000306244 7001_ $$aLilly, Jeremiah$$b18
000306244 7001_ $$aBorisova, Ekaterina$$b19
000306244 7001_ $$aAmbrozkiewicz, Mateusz C$$b20
000306244 7001_ $$aTarabykin, Victor$$b21
000306244 7001_ $$aKupiec, Martin$$b22
000306244 7001_ $$aThaker, Maulik$$b23
000306244 7001_ $$aRotin, Daniela$$b24
000306244 7001_ $$aPrag, Gali$$b25
000306244 773__ $$0PERI:(DE-600)1355192-9$$a10.1016/j.jmb.2021.167276$$gVol. 433, no. 23, p. 167276 -$$n23$$p167276$$tJournal of molecular biology$$v433$$x0022-2836$$y2021
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