In Vitro Analysis of E3 Ubiquitin Ligase Function

Summary

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.

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~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.

Introduction

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....

Protocol

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 (Na₂HPO₄.2 H₂O), and 20 mM of potassium-dihydrogen phosphate (KH₂PO₄) in 1 L of double-distilled H₂O (ddH

Results

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........

Discussion

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.......

Materials

List of materials used in this article

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