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)

Abstract

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
PublisherElsevier
Chapter12
Pages257-280
Number of pages24
Volume618
ISBN (Print)9780128163597
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Enzymology
PublisherElsevier
Volume618
ISSN (Print)0076-6879

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Keywords

  • 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

Cite this

Branigan, E., Plechanovová, A., & Hay, R. T. (2019). Methods to analyze STUbL activity. In M. Hochstrasser (Ed.), Methods in Enzymology (Vol. 618, pp. 257-280). (Methods in Enzymology; Vol. 618). Elsevier. https://doi.org/10.1016/bs.mie.2018.11.005