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.
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.
1. Plasmid Construction
2. Virus Production
Culture medium – DMEM (HEK 293FT)
DMEM
10% FBS
0.1 mM MEM NEAA
6 mM L-glutamine
1 mM MEM Sodium Pyruvate
1% Pen-Strep (optional)
500 μg/ml Geneticin (optional)
3. Virus Concentration by Polyethylene Glycol (PEG) Precipitation
4. Titering of Virus
Culture medium – DMEM (HFF-2/U2-OS)
DMEM
10% FBS
6mM L-glutamine
1% Pen-Strep
5. Transduction of Human Diploid Fibroblasts (This procedure can be used for any cell type.)
6. Measurement by Flow Cytometry
7. Representative Results
The GFP-dgn fusion protein carries a sequence which is targeted to the proteasome and therefore the protein is immediately degraded; it corresponds to the decrease in GFP fluorescence signal. The frameshift mutant (GFP-dgnFS) carries a mutated version of this sequence and is not degraded by proteasome; it leads to the higher green fluorescence. For these reasons, young HDFs with expected high proteasome activity and transduced with GFP-dgn show a low (6% positive cells) fluorescence signal in both flow cytometry measurement and in epifluorescence (Figure 2A and B, Figure 3). The same HDFs transduced with GFP-dgnFS display 39.7% of positive cells. The treatment of the cells with proteasome inhibitor LLnL (N-acetyl-L-leucyl-L-leucyl-L-norleucinal) raised the signal to maximum of 62.9% in both, GFP-dgn and GFP-dgnFS cells (Figure 2A and B).
To determine possible decline in proteasome activity in aged human skin samples, dermal fibroblasts isolated from young, middle-aged and old donors were infected with GFP-dgn and GFP-dgnFS, as described above and cultivated to the same passage number before analysis by flow cytometry. In these experiments, a clear-cut increase in GFP signal between young individuals (11.2±0.88% GFP-positive cells) and middle-aged donors (20.4±2.27% GFP-positive cells, P=0.003) has been observed, indicating a decrease in proteasome activity in samples obtained from aged donors7 (Figure 4). No further decrease in proteasome activity was observed in fibroblasts isolated from oldest individuals (Figure 4). Fluorescence intensity for GFP-dgnFS was in all cases ~90% (data not shown) which indicates a high transfection efficiency for the three different age groups.
Figure 1. The GFP-dgn and GFP-dgnFS sequence. Displayed is the 3′-end of GFP (green) the multiple cloning site of the pEGFP-CL1 vector (grey) and the dgn/dgnFS sequence (red).
Figure 2. Flow cytometry analysis of GFP-dgn and GFP-dgnFS human diploid fibroblasts. A. Young human foreskin fibroblasts (HFF-2) were infected with lentiviral vectors carrying a green fluorescent protein (GFP)–dgn gene or a GFP–dgnFS (frameshift) construct, as indicated and analyzed for GFP fluorescence using flow cytometry (FACS Canto II, Becton Dickinson). Where indicated, cells were also treated for 3h with proteasome inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Uninfected cells were used as a control. Experiments were performed in triplicates. B. Data are representative of three independent experiments. The numbers reflect the amount of GFP positive cells. Click here to view larger figure.
Figure 3. Fluorescence microscopy analysis of GFP-dgn and GFP-dgnFS HFF-2 cells.
Young HFF-2 were treated as in Figure 2. At 9 days after infection, cells were visualized by fluorescence and phase contrast microscopy.
Figure 4. Changes in proteasome activity in human skin aging.
The human foreskin fibroblasts from nine different donors in the indicated age groups were minimally expanded and infected with lentiviral constructs coding for GFP–dgn. At 9 days after infection, the cells were analyzed by flow cytometry. Data were obtained on three samples per age group in duplicates (± SE).
Figure 5. Experimental approach. Custom-oligo nucleotides for dgn and dgnFS are cloned into the pEGFP-C1 vector and viruses are produced for each construct using HEK 293FT cells. The titer of the viruses is determined. Cells are transduced with the virus and expanded. After the favored treatment the cells are analyzed for fluorescence signal by flow cytometry.
Figure 6. Map of pLenti GFP-dgn. The map of the pLenti6/V5-DEST vector including the GFP-dgn sequence is displayed. Abbreviations: PCMV (CMV promoter), GFP-dgn (sequence of GFP-dgn), PSV40 (SV40 early promoter), EM7 (EM7 promoter), Blasticidin (Blasticidin resistance gene), ΔU3/3’LTR (3’LTR with deleted U3 region), SV40 pA (SV40 polyadenylation signal), Ampicillin (Ampicillin resistance gene), pUC ori (pUC origin), PRSV/5’LTR (RSV/5’LTR hybrid promoter), Ψ (HIV-1 Ψ packaging signal), RRE (HIV-1 Rev response element).
Figure 7. U2-OS titering plate. U2-OS were seeded out on a 6-well plate and transfected with different virus concentrations (dilution of 1/100 and 1/10, 1, 5 and 10 μl of concentrated viral supernatant). One well was used as untransfected control (NT). After 6-7 days, the cells were stained with crystal violet and air dried.
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-destabilized GFP reporter as used in this publication 14.
Unfortunately, GFP as a reporter has some imperfection which has been shown by Dantuma et al. and Bowman et al. 12,15. First, destabilized GFP can accumulate without the inhibition of the proteasome due to unknown reasons. As the ubiquitin-proteasome machinery is a system whose functionality is dependent on multiple steps, a disturbance prior to the degradation can lead to the accumulation of the reporter substrate and result in false-positive effects 12. Second, GFP-based reporter substrates are due to their short half-lives sensitive to changes in transcription and translation. Any change in GFP fluorescence can therefore be a result of reduced or increased synthesis and translation 15. These problems have been addressed in other studies by modifications of GFP as follows:
Proteasome activity was found to be decreased in cellular senescence and organismal aging 7, 8, 9, 10. In the field of aging research, the focus is mainly on the removal of oxidized proteins where the proteasome plays a key role, and there is no conclusive evidence that protein ubiquitylation precedes degradation in this case 2. Previous work has also shown that the use of GFP with a mutated uncleavable ubiquitin moiety is suitable to study the complex process of protein degradation 16. This GFP-construct gives the possibility to monitor proteasome activity without the potential influence to its activity via disturbances in the ubiquitin machinery.
In this work, we used a degron-destabilized GFP-based reporter protein (GFP-dgn) to monitor ubiquitin-proteasome activity in living human diploid fibroblasts. To have an overview of the transfection efficiency, the functionality and the transcription/translation levels of the GFP-dgn reporter, several appropriate controls were included. We used young untreated GFP-dgn transfected fibroblasts to show that there is almost no green fluorescence visible and proteasome function is not affected. To validate, that the signal increases with the inhibition of the proteasome, we treated GFP-dgn transfected fibroblasts with a proteasome inhibitor to see the accumulation of GPF after inhibition. As a third control we used GFP-dgnFS. With this construct we can show the overall transfection efficiency which can differ from cell strain to cell strain and with all three controls we have an overview over the synthesis rate of the constructs within the cell.
The method presented here enables the user to measure easily and rapidly the ubiquitin-proteasome activity in living cells. A stable overexpression of the GFP reporter substrate can be achieved with different techniques 17. In this publication, transfection of the GFP-dgn cDNA into the cells is achieved by lentiviral system which provides a stable expression. Additionally, the use of this system enables high transfection efficiency independent of the cell type, cell metabolic condition or donor age 7. We have successfully used this protocol to introduce DNA to the endothelial cells in which other methods of transfection failed 18. Nevertheless, previous experiments showed that cells that are expanded longer in culture display a lower fluorescence signal than freshly transfected cells possibly due to the longer selection pressure.
The PEG precipitation is used to increase the titer of the transforming units per ml (TU/ml). If good titers are reached (above 5×105 TU/ml), the PEG precipitation can be omitted. An important step during PEG precipitation is to avoid vigorous suspending or pipetting which generates air bubbles. It can inactivate the virus particles and significantly decrease transfection efficiency.
When using proteasome inhibitors a priori experiments should be performed. The final concentration used and incubation time are cell type dependent and have to be determined experimentally. Previous observations on HFF-2 cells revealed that 3h incubation with LLnL should not be exceeded because of 1) the toxic effects of the inhibitor 2) too high GFP signal that can be reached, or 3) side effects of the proteasome inhibition over longer time periods.
The authors have nothing to disclose.
This study was funded by: National Research Network on Aging (NFN S93) by the Austrian Science Foundation (FWF), European Commission Integrated Projects MiMAGE and PROTEOMAGE, Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NGI/NWO; 05040202 and 050-060-810 NCHA), the EU funded Network of Excellence Lifespan (FP6 036894), and Innovation Oriented Research Program on Genomics (SenterNovem; IGE01014 and IGE5007).
Name of the reagent | Company | Catalogue number |
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 | D5546 |
PVDF filter (Rotilabo-Spritzenfilter) | Roth | P667.1 |
Polyethylene glycol | Sigma | P2139 |
NaCl | Merck | 1.06404.1000 |
Dulbecco’s Phosphate Buffered Saline 1x (PBS) | Invitrogen | 14190 |
hexadimethrine bromide | Sigma | 10,768-9 |
Blasticidin | Invitrogen | R21001 |
Crystal violet | Sigma | 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 | G8769 |
Geneticin | Invitrogen | 11811023 |
CaCl2 | Merck | C5080 |
Hepes | Sigma | H3375 |
Trypsin-EDTA (0.05%) | Invitrogen | 25300054 |