Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Summary

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Abstract

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Protocol

1. Plasmid Construction

1. Order custom oligo-nucleotides encoding for dgn (ACKNWFSSLSHFVIHL¹¹) and for dgnFS (HARTGSLACPTSSSICE) and ligate it into the pEGFP-C1 vector to obtain the fusion of the GFP with dgn/dgnFS (Figure 1).
2. Amplify the coding sequence for GFP-dgn and GFP-dgnFS by PCR according to the protocol of the pENTR Directional TOPO Cloning Kit and continue with the pLenti6/V5 Directional TOPO Cloning Kit (Figure 6).

2. Virus Production

1. The day before transfection (Day 1) seed out HEK 293FT cells in a T75 flasks so that they will be 90-95% con....

Discussion

The first publication using green fluorescent protein (GFP) as a reporter substrate for ubiquitin-proteasome activity was published in 2000 ¹². Since then, GFP has become a common tool to visualize cellular activities, especially the ubiquitin-proteasome process. To monitor ubiquitin-proteasome activity in vivo a transgenic mouse model with a GFP-based reporter has been introduced ¹³. Additional in vivo research established another transgenic mouse model with a similar degron-desta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pEGFP-C1 Vector	BD Bioscience Clontech	6084-1
pENTR Directional TOPO Cloning Kit	Invitrogen	K2400-20
pLenti6/V5 Directional TOPO Cloning Kit	Invitrogen	V496-10
Lipofectamine 2000 Reagent	Invitrogen	11668019
DMEM	Sigma-Aldrich	D5546
PVDF filter (Rotilabo-Spritzenfilter)	Carl Roth Gmbh	P667.1
Polyethylene glycol	Sigma-Aldrich	P2139
NaCl	Merck & Co., Inc.	1.06404.1000
Dulbecco's Phosphate Buffered Saline 1x (PBS)	Invitrogen	14190
hexadimethrine bromide	Sigma-Aldrich	10,768-9
Blasticidin	Invitrogen	R21001
Crystal violet	Sigma-Aldrich	C3886
FACS tubes	BD Biosciences
Penicillin Streptomycin (Pen-Strep)	Invitrogen	15140130
L-glutamine 200 mM	Invitrogen	25030024
Fetal Bovine Serum (FBS)	Biochrom AG	S0115
MEM Non-Essential Amino Acids (NEAA) 100x	Invitrogen	11140035
MEM Sodium Pyruvate 100 mM	Invitrogen	11360039
D-(+)-Glucose (45%)	Sigma-Aldrich	G8769
Geneticin	Invitrogen	11811023
CaCl2	Merck & Co., Inc.	C5080
Hepes	Sigma-Aldrich	H3375
Trypsin-EDTA (0.05%)	Invitrogen	25300054