Universität zu Köln

Muller, Leonie, Kutzner, Carl Elias ORCID: 0000-0003-4053-2231, Balaji, Vishnu and Hoppe, Thorsten (2021). In Vitro Analysis of E3 Ubiquitin Ligase Function. J. Vis. Exp. (171). CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS. ISSN 1940-087X

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Abstract

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2 similar to Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Muller, Leonie UNSPECIFIED UNSPECIFIED UNSPECIFIED Kutzner, Carl Elias UNSPECIFIED orcid.org/0000-0003-4053-2231 UNSPECIFIED Balaji, Vishnu UNSPECIFIED UNSPECIFIED UNSPECIFIED Hoppe, Thorsten UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-565658
DOI:	10.3791/62393
Journal or Publication Title:	J. Vis. Exp.
Number:	171
Date:	2021
Publisher:	JOURNAL OF VISUALIZED EXPERIMENTS
Place of Publication:	CAMBRIDGE
ISSN:	1940-087X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CHIP; COMPLEX; MECHANISM; REVEALS; ENZYMES; E4 Multiple languages Multidisciplinary Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/56565