Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Summary

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Abstract

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

Introduction

Post-translational modifications (PTMs) are an important mechanism regarding protein regulation, which is essential for cell homeostasis. Protein ubiquitylation is a dynamic and intricate modification that creates an assortment of different signals resulting in several cellular outcomes in eukaryotic organisms. Ubiquitylation is a reversible process consisting in the attachment of a ubiquitin protein containing 76 amino acids to the substrate, occurring in an enzymatic cascade composed by three distinct reactions¹. The first step is characterized by ubiquitin activation, which depends on an ATP hydrolysis to form a high-energy thioester-linked ....

Protocol

NOTE: An overview of ubiquitylation assay protocol in mammalian cells is represented in Figure 1.

Figure 1. Overview of the ubiquitylation assay procedure. Please click here to view a larger version.......

Discussion

Ubiquitylation is an essential post-translational modification that regulates the levels of several proteins and plays a crucial role in many signaling pathways and biological processes, ensuring a healthy intracellular environment. The ubiquitin-proteasome system (UPS) is one of the main focuses of recent pharmaceutical research, providing the possibility of stabilizing tumor suppressors or inducing the degradation of oncogenic products²². For instance, the aberrant proliferation of plasma cell n.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microtube	Axygen	PMI110-06A
100 mm TC-treated culture dish	Corning	430167
15 mL tube	Corning	430766
96-well plate	Cralplast	655111
Agarose-anti-HA beads	Sigma-Aldrich	E6779
Anti Mouse antibody	Seracare	5220-0341	Goat anti-Mouse IgG
Anti Rabbit antibody	Seracare	5220-0337	Goat anti-Rabbit IgG
Anti-Actin antibody	Sigma-Aldrich	A3853	Dilution used: 1:2000
Anti-Fbxo7 antibody	Sigma-Aldrich	SAB1407251	Dilution used: 1:1000
Anti-HA antibody	Sigma-Aldrich	H3663	Dilution used: 1:1000
Anti-Myc antibody	Cell Signalling	2272	Dilution used: 1:1000
Bradford reagent	Sigma-Aldrich	B6916-500ML
BSA	Sigma-Aldrich	A9647-100G	Bovine Serum Albumin
Cell incubator	Nuaire	NU-4850
Centrifuge	Eppendorf	5804R	500 x g for 5 min
ChemiDoc	BioRad
Digital pH meter	Kasvi	K39-2014B
Dulbecco’s Modified Eagle’s Medium	Corning	10-017-CRV	High glucose
Fetal bovine serum	Gibco	F4135	Filtrate prior use
HA peptide	Sigma-Aldrich	I2149
HEK293T cells	ATCC	CRL-3216
Hepes	Gibco	15630080
KCl	VWR Life Science	0365-500G
Kline rotator	Global Trade Technology	GT-2OIBD
MG-132	Boston Biochem	I-130
Microcentrifuge	Eppendorf	5418R
Na3VO4 (Ortovanadato)
NaF
Nitrocellulose blotting membrane	GE Healthcare	10600016
NP40 (IGEPAL CA-630)	Sigma-Aldrich	I8896-100ML
Optical microscope	OPTIKA microscopes	SN510768
Opti-MEM	Gibco	31985-070
pcDNA3	Invitrogen	V79020	For mammalian expression
pcDNA3-2xFlag-Fbxo7	Kindly donated by Dr. Marcelo Damário		Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI
pcDNA3-2xFlag-Fbxo7-ΔF-box	Kindly donated by Dr. Marcelo Damário		Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. Δ335-367
pcDNA3-UXTV2-HA	Kindly donated by Dr. Marcelo Damário		Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI
pCMV-6xHis-Myc-Ubiquitin	Kindly donated by Dr. Marcelo Damário		Tag 6x-His-Myc (N-terminal). Restriction enzymes: EcoRI and KpnI
Pen Strep Glutamine 100x	Gibco	10378-016
Phosphate buffered saline 10x	AccuGENE	51226	To obtain a 1x PBS, dilute the 10x PBS into ultrapure water
Polyethylenimine (PEI)	Sigma-Aldrich	9002-98-6
Ponceau S	VWR Life Science	0860-50G
Protease inhibitor cocktail SIGMAFAST	Sigma-Aldrich	S8820
Rocking Shaker	Kasvi	19010005
SDS-PAGE system	BioRad	165-8004
Solution Homogenizer	Phoenix Luferco	AP-22
Trizma base	Sigma-Aldrich	T6066-500G
Trypsine (TrypLe Express)	Gibco	12605-028
Western Blotting Luminol Reagent	Santa Cruz Biotechnology	SC-2048