TY  - JOUR
AU  - Levin-Kravets, Olga
AU  - Kordonsky, Alina
AU  - Shusterman, Anna
AU  - Biswas, Sagnik
AU  - Persaud, Avinash
AU  - Elias, Sivan
AU  - Langut, Yael
AU  - Florentin, Amir
AU  - Simpson-Lavy, Kobi J
AU  - Yariv, Elon
AU  - Avishid, Reut
AU  - Sror, Mor
AU  - Almog, Ofir
AU  - Marshanski, Tal
AU  - Kadosh, Shira
AU  - Ben David, Nicole
AU  - Manori, Bar
AU  - Fischer, Zohar
AU  - Lilly, Jeremiah
AU  - Borisova, Ekaterina
AU  - Ambrozkiewicz, Mateusz C
AU  - Tarabykin, Victor
AU  - Kupiec, Martin
AU  - Thaker, Maulik
AU  - Rotin, Daniela
AU  - Prag, Gali
TI  - Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.
JO  - Journal of molecular biology
VL  - 433
IS  - 23
SN  - 0022-2836
CY  - Amsterdam [u.a.]
PB  - Elsevier
M1  - DKFZ-2025-02473
SP  - 167276
PY  - 2021
N1  - #DKFZ-MOST-Ca196#
AB  - 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.
KW  - Biological Assay: methods
KW  - Chloramphenicol O-Acetyltransferase: genetics
KW  - Chloramphenicol O-Acetyltransferase: metabolism
KW  - Enzyme Activation
KW  - Escherichia coli: genetics
KW  - Escherichia coli: metabolism
KW  - Gene Expression
KW  - Genes, Reporter
KW  - Protein Processing, Post-Translational
KW  - Proteolysis
KW  - Ubiquitin-Protein Ligases: metabolism
KW  - Ubiquitination
KW  - Kaufman oculocerebrofacial syndrome (Other)
KW  - protein-protein interaction assay (Other)
KW  - ubiquitylation (Other)
KW  - Chloramphenicol O-Acetyltransferase (NLM Chemicals)
KW  - Ubiquitin-Protein Ligases (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:34599943
DO  - DOI:10.1016/j.jmb.2021.167276
UR  - https://inrepo02.dkfz.de/record/306244
ER  -