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  1. Artikel ; Online: Exploring ligand interactions with human phosphomannomutases using recombinant bacterial thermal shift assay and biochemical validation.

    Monticelli, Maria / Hay Mele, Bruno / Wright, Demi Marie / Guerriero, Simone / Andreotti, Giuseppina / Cubellis, Maria Vittoria

    Biochimie

    2024  Band 222, Seite(n) 123–131

    Abstract: PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of ...

    Abstract PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches. We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding.
    Sprache Englisch
    Erscheinungsdatum 2024-03-06
    Erscheinungsland France
    Dokumenttyp Journal Article
    ZDB-ID 120345-9
    ISSN 1638-6183 ; 0300-9084
    ISSN (online) 1638-6183
    ISSN 0300-9084
    DOI 10.1016/j.biochi.2024.02.011
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  2. Artikel ; Online: ReBaTSA: A simplified CeTSA protocol for studying recombinant mutant proteins in bacterial extracts.

    Monticelli, Maria / Wright, Demi Marie / Cubellis, Maria Vittoria / Andreotti, Giuseppina

    Biochimica et biophysica acta. General subjects

    2023  Band 1868, Heft 2, Seite(n) 130526

    Abstract: Introduction: The study of protein stability is crucial to biochemistry and relies on different methodologies. Recently, the Cellular Thermal Shift Assay has been introduced to study protein stability in whole cells.: Methods: We report a novel ... ...

    Abstract Introduction: The study of protein stability is crucial to biochemistry and relies on different methodologies. Recently, the Cellular Thermal Shift Assay has been introduced to study protein stability in whole cells.
    Methods: We report a novel application of CeTSA named ReBaTSA. This Recombinant Bacterial TSA was performed using clear extracts from bacteria expressing a recombinant protein, incubated at different temperatures, centrifuged and analyzed via SDS-PAGE.
    Results and conclusions: We demonstrated the feasibility and reliability of this simplified approach. We validated the method using the protein phosphomannomutase-2 and its common mutants, which were compared in the presence or the absence of a known ligand.
    Mesh-Begriff(e) Bacterial Lysates ; Mutant Proteins ; Reproducibility of Results ; Protein Stability ; Recombinant Proteins/genetics
    Chemische Substanzen Bacterial Lysates ; Mutant Proteins ; Recombinant Proteins
    Sprache Englisch
    Erscheinungsdatum 2023-12-03
    Erscheinungsland Netherlands
    Dokumenttyp Journal Article
    ZDB-ID 60-7
    ISSN 1872-8006 ; 1879-2596 ; 1879-260X ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    ISSN (online) 1872-8006 ; 1879-2596 ; 1879-260X ; 1879-2642 ; 1879-2618 ; 1879-2650
    ISSN 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    DOI 10.1016/j.bbagen.2023.130526
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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