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In Vitro Analysis of E3 Ubiquitin Ligase Function

Published: May 14th, 2021



1Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), University of Cologne, 2Center for Molecular Medicine Cologne, University of Cologne

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

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~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.

Ubiquitylation is the process by which Ub is covalently linked to a substrate protein1. The Ub modification is catalyzed by successive enzymatic reactions involving the action of three different enzyme classes, i.e., Ub-activating enzymes (E1s), Ub-conjugating enzymes (E2s), Ub ligases (E3s), and possibly Ub chain elongation factors (E4s)2,3,4,5. After adenosine triphosphate (ATP)- and magnesium (Mg2+)-dependent activation of Ub by E1, the active site cysteine of E1 attacks the C-terminal glycine o....

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1. Preparation of buffers and reagents

NOTE: Buffers and reagents that were manually prepared in the laboratory are listed below. All other buffers and reagents used in the protocols were purchased from different sources and used according to the manufacturers' instructions.

  1. Prepare 10x phosphate-buffered saline (10x PBS). For this purpose, mix 1.37 M sodium chloride (NaCl), 27 mM potassium chloride (KCl), 80 mM of disodium-hydrogen-phosphate dihydrate (Na2HPO4<.......

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To identify E2 enzymes that cooperate with the ubiquitin ligase CHIP, a set of E2 candidates was tested in individual in vitro ubiquitylation reactions. Cooperating E2-E3 pairs were monitored by the formation of E3-dependent ubiquitylation products, i.e., auto-ubiquitylation of the E3 ligase and the formation of free Ub polymers. The ubiquitylation products were analyzed by western blotting. Data interpretation is based on the size comparison of the resulting protein bands with molecular weight markers........

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This paper describes basic in vitro ubiquitylation methods for the analysis of E3 ligase function. When performing in vitro ubiquitylation assays, it should be considered that some E2 enzymes can perform auto-ubiquitylation owing to the attack of their active cysteine on their own lysine residues that are located in close proximity to the active site30. To circumvent this problem, the use of an E2 mutant is recommended in which the respective lysine residue is exchanged for argin.......

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We thank the members of our laboratory for critical discussion and helpful advice on the manuscript. We apologize for not having cited valuable contributions due to size limitation. This work is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - SFB 1218 - Projektnumber 269925409 and Cluster of Excellence EXC 229/ CECAD to TH. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy - EXC 2030 - 390661388 and - SFB 1218 - Projektnumber 269925409 to T.H. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) im Rahmen der deutschen Exzellenzstrateg....

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Name Company Catalog Number Comments
Amershan Protran 0.1 µm NC GE Healthcare 10600000 nitrocellulose membrane
Anti-CHIP Cell Signaling 2080 Monoclonal rabbit anti-CHIP antibody, clone C3B6
Anti-MYC Roche OP10 Monoclonal mouse anti-MYC antibody, clone 9E10
Anti-ubiquitin Upstate 05-944 Monoclonal mouse anti-Ub antibody, clone P4D1-A11
Apyrase Sigma A6535-100UN
ATP (10x) Enzo 12091903
BSA Sigma A6003-10G
EDTA Roth 8043.2
KCl Roth 6781.1
K2HPO4 Roth P749.2
KH2PO4 Roth 3904.1
LDS sample buffer (4x) novex B0007
L-Lysine Sigma L5501-5G
MES Roth 4256.4
MeOH VWR Chemicals 2,08,47,307 100%
Milchpulver Roth T145.3
NaCl Roth P029.3
NuPAGE Antioxidant invitrogen NP0005
NuPAGE Transfer buffer (20x) novex NP0006-1
Page ruler plus Thermo Fisher 26619 Protein ladder
RotiBlock Roth A151.1 Blocking reagent
SDS (20%) Roth 1057.1
S1000 Thermal Cycler Bio Rad 1852196
Trans-Blot Turbo Bio Rad 1704150EDU Transfer system
Tris base Roth 4855.3
Tween 20 Roth 9127.2
UbcH Enzyme Set BostonBiochem K-980B E2 enzymes
Ubiquitin BostonBiochem U-100H
Ubiquitin-activating enzyme E1 Enzo BML-UW941U-0050
Ubiquitylation buffer (10x) Enzo BML-KW9885-001
Whatman blotting paper Bio Rad 1703969 Extra thick filter paper

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