Methods to analyze STUbL activity

Emma Branigan, Anna Plechanovová, Ronald T. Hay (Lead / Corresponding author)

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

    3 Citations (Scopus)


    Posttranslational modification with small ubiquitin-like modifier (SUMO) plays an important role in many biological processes. SUMO-targeted ubiquitin E3 ligases (STUbLs) are part of the really interesting new gene (RING)-type family of ubiquitin E3 ligases. STUbLs recognize their SUMO-modified substrates via SUMO-interaction motifs and ubiquitinate them via the RING domain. As a result, they form a link between the ubiquitin and SUMO signaling pathways. STUbL activity is required for the maintenance of genome stability, the repair of damaged DNA and to target SUMO-modified proteins for degradation by the proteasome. In vitro assays for STUbL activity have been developed and used to identify their cognate ubiquitin-conjugating enzymes (E2s), to determine their substrate requirements, and to characterize the types of ubiquitin chains linked to substrates. While we have focused on the STUbL RING finger protein 4 (RNF4) the methods we describe can be extended to other STUbLs. We also describe an assay for RNF4 ubiquitination activity based on fluorescence polarization, suitable for high-throughput compound screening in drug discovery.

    Original languageEnglish
    Title of host publicationMethods in Enzymology
    EditorsMark Hochstrasser
    Place of PublicationCambridge, MA
    Number of pages24
    ISBN (Print)9780128163597
    Publication statusPublished - 2019

    Publication series

    NameMethods in Enzymology
    ISSN (Print)0076-6879


    • Fluorescence polarization
    • Fluorescent labeling
    • High-throughput screening
    • Quantitation
    • RING (really interesting new gene)
    • RNF4 (RING finger protein 4)
    • STUbL (SUMO-targeted ubiquitin E3 ligase)
    • Ubiquitination

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology


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