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  1. Article: Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase

    Fottner, Maximilian / Heimgärtner, Johannes / Gantz, Maximilian / Mühlhofer, Rahel / Nast-Kolb, Timon / Lang, Kathrin

    Journal of the American Chemical Society. 2022 July 18, v. 144, no. 29

    2022  

    Abstract: Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic ...

    Abstract Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEP-mediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitin- and ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region.
    Keywords Lewis bases ; genetic code ; moieties ; peptides ; point mutation ; proteinases ; therapeutics
    Language English
    Dates of publication 2022-0718
    Size p. 13118-13126.
    Publishing place American Chemical Society
    Document type Article
    ZDB-ID 3155-0
    ISSN 1520-5126 ; 0002-7863
    ISSN (online) 1520-5126
    ISSN 0002-7863
    DOI 10.1021/jacs.2c02191
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

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  2. Article ; Online: Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site-Specific Photocrosslinking Approach.

    Nguyen, Tuan-Anh / Gronauer, Thomas F / Nast-Kolb, Timon / Sieber, Stephan A / Lang, Kathrin

    Angewandte Chemie (International ed. in English)

    2022  Volume 61, Issue 10, Page(s) e202111085

    Abstract: Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human ... ...

    Abstract Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Site-specific incorporation of the diazirine-bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV-irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis. In addition to confirming many of the previously annotated hClpP substrates, our approach adds a diverse set of new proteins to the hClpP interactome. Importantly, our workflow allows identifying substrate dynamics upon application of external cues in an unbiased manner. Identification of unique hClpP-substrate proteins upon induction of mt oxidative stress, suggests that hClpP counteracts oxidative stress by processing of proteins that are involved in respiratory chain complex synthesis and maturation as well as in catabolic pathways.
    MeSH term(s) Cross-Linking Reagents/chemistry ; Cross-Linking Reagents/metabolism ; Endopeptidase Clp/chemistry ; Endopeptidase Clp/metabolism ; Humans ; Mitochondria/enzymology ; Models, Molecular ; Molecular Structure ; Photochemical Processes ; Substrate Specificity
    Chemical Substances Cross-Linking Reagents ; ClpP protein, human (EC 3.4.21.92) ; Endopeptidase Clp (EC 3.4.21.92)
    Language English
    Publishing date 2022-01-14
    Publishing country Germany
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2011836-3
    ISSN 1521-3773 ; 1433-7851
    ISSN (online) 1521-3773
    ISSN 1433-7851
    DOI 10.1002/anie.202111085
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase.

    Fottner, Maximilian / Heimgärtner, Johannes / Gantz, Maximilian / Mühlhofer, Rahel / Nast-Kolb, Timon / Lang, Kathrin

    Journal of the American Chemical Society

    2022  Volume 144, Issue 29, Page(s) 13118–13126

    Abstract: Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic ...

    Abstract Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEP-mediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitin- and ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region.
    MeSH term(s) Cysteine Endopeptidases/metabolism ; Peptides ; Recombinant Proteins/metabolism ; Ubiquitin/genetics
    Chemical Substances Peptides ; Recombinant Proteins ; Ubiquitin ; Cysteine Endopeptidases (EC 3.4.22.-) ; asparaginylendopeptidase (EC 3.4.22.34)
    Language English
    Publishing date 2022-07-18
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 3155-0
    ISSN 1520-5126 ; 0002-7863
    ISSN (online) 1520-5126
    ISSN 0002-7863
    DOI 10.1021/jacs.2c02191
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    In stock of ZB MED Bonn / Germany

    Z 4' 55/32: Show issues
    Z 7.3: Show issues

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

    Inter-library loan at ZB MED

    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

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