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  1. AU="Manori, Bar"
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  1. Article ; Online: Chloride intracellular channel (CLIC) proteins function as fusogens.

    Manori, Bar / Vaknin, Alisa / Vaňková, Pavla / Nitzan, Anat / Zaidel-Bar, Ronen / Man, Petr / Giladi, Moshe / Haitin, Yoni

    Nature communications

    2024  Volume 15, Issue 1, Page(s) 2085

    Abstract: Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our ...

    Abstract Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.
    MeSH term(s) Animals ; Chlorides/metabolism ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans/metabolism ; Chloride Channels/metabolism ; Liposomes
    Chemical Substances Chlorides ; Chloride Channels ; Liposomes
    Language English
    Publishing date 2024-03-07
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-024-46301-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

    Levin-Kravets, Olga / Kordonsky, Alina / Shusterman, Anna / Biswas, Sagnik / Persaud, Avinash / Elias, Sivan / Langut, Yael / Florentin, Amir / Simpson-Lavy, Kobi J. / Yariv, Elon / Avishid, Reut / Sror, Mor / Almog, Ofir / Marshanski, Tal / kadosh, Shira / Ben David, Nicole / Manori, Bar / Fischer, Zohar / Lilly, Jeremiah /
    Borisova, Ekaterina / Ambrozkiewicz, Mateusz C. / Tarabykin, Victor / Kupiec, Martin / Thaker, Maulik / Rotin, Daniela / Prag, Gali

    Journal of molecular biology. 2021 Nov. 19, v. 433, no. 23

    2021  

    Abstract: 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, ... ...

    Abstract 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.
    Keywords Escherichia coli ; chloramphenicol acetyltransferase ; genetic selection ; humans ; metabolites ; molecular biology ; oligomerization ; sowing ; ubiquitin ; yeasts
    Language English
    Dates of publication 2021-1119
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2021.167276
    Database NAL-Catalogue (AGRICOLA)

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  3. Article ; Online: Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

    Levin-Kravets, Olga / Kordonsky, Alina / Shusterman, Anna / Biswas, Sagnik / Persaud, Avinash / Elias, Sivan / Langut, Yael / Florentin, Amir / Simpson-Lavy, Kobi J / Yariv, Elon / Avishid, Reut / Sror, Mor / Almog, Ofir / Marshanski, Tal / Kadosh, Shira / Ben David, Nicole / Manori, Bar / Fischer, Zohar / Lilly, Jeremiah /
    Borisova, Ekaterina / Ambrozkiewicz, Mateusz C / Tarabykin, Victor / Kupiec, Martin / Thaker, Maulik / Rotin, Daniela / Prag, Gali

    Journal of molecular biology

    2021  Volume 433, Issue 23, Page(s) 167276

    Abstract: 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, ... ...

    Abstract 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.
    MeSH term(s) Biological Assay/methods ; Chloramphenicol O-Acetyltransferase/genetics ; Chloramphenicol O-Acetyltransferase/metabolism ; Enzyme Activation ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression ; Genes, Reporter ; Protein Processing, Post-Translational ; Proteolysis ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination
    Chemical Substances Chloramphenicol O-Acetyltransferase (EC 2.3.1.28) ; Ubiquitin-Protein Ligases (EC 2.3.2.27)
    Language English
    Publishing date 2021-09-29
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2021.167276
    Database MEDical Literature Analysis and Retrieval System OnLINE

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