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

Fingerprint

Ubiquitin-Protein Ligases
Ubiquitin
Genes
Assays
Substrates
Ubiquitin-Conjugating Enzymes
Biological Phenomena
Proteins
Fluorescence Polarization
Genomic Instability
Ubiquitination
Proteasome Endopeptidase Complex
Drug Discovery
Post Translational Protein Processing
DNA Repair
Proteolysis
Screening
Repair
Fluorescence
Maintenance

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). Cambridge, MA: Elsevier. https://doi.org/10.1016/bs.mie.2018.11.005
Branigan, Emma ; Plechanovová, Anna ; Hay, Ronald T. / Methods to analyze STUbL activity. Methods in Enzymology. editor / Mark Hochstrasser. Vol. 618 Cambridge, MA : Elsevier, 2019. pp. 257-280 (Methods in Enzymology).
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Branigan, E, Plechanovová, A & Hay, RT 2019, Methods to analyze STUbL activity. in M Hochstrasser (ed.), Methods in Enzymology. vol. 618, Methods in Enzymology, vol. 618, Elsevier, Cambridge, MA, pp. 257-280. https://doi.org/10.1016/bs.mie.2018.11.005

Methods to analyze STUbL activity. / Branigan, Emma; Plechanovová, Anna; Hay, Ronald T. (Lead / Corresponding author).

Methods in Enzymology. ed. / Mark Hochstrasser. Vol. 618 Cambridge, MA : Elsevier, 2019. p. 257-280 (Methods in Enzymology; Vol. 618).

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

TY - CHAP

T1 - Methods to analyze STUbL activity

AU - Branigan, Emma

AU - Plechanovová, Anna

AU - Hay, Ronald T.

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PY - 2019

Y1 - 2019

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AB - 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.

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KW - Fluorescent labeling

KW - High-throughput screening

KW - Quantitation

KW - RING (really interesting new gene)

KW - RNF4 (RING finger protein 4)

KW - STUbL (SUMO-targeted ubiquitin E3 ligase)

KW - Ubiquitination

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U2 - 10.1016/bs.mie.2018.11.005

DO - 10.1016/bs.mie.2018.11.005

M3 - Chapter (peer-reviewed)

C2 - 30850055

AN - SCOPUS:85059345076

SN - 9780128163597

VL - 618

T3 - Methods in Enzymology

SP - 257

EP - 280

BT - Methods in Enzymology

A2 - Hochstrasser, Mark

PB - Elsevier

CY - Cambridge, MA

ER -

Branigan E, Plechanovová A, Hay RT. Methods to analyze STUbL activity. In Hochstrasser M, editor, Methods in Enzymology. Vol. 618. Cambridge, MA: Elsevier. 2019. p. 257-280. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2018.11.005