In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.
This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).
1. Seeding the cells
2. Transfection
3. Splitting the cells
4. Heat shock
5. Cell lysis and luciferase assay
6. Representative Results
Protein refolding is directly related to the time of recovery therefore to test if the system is working properly, an assay has to be performed collecting the cells at different time points. A direct correlation time/percentage of refolding indicates that the experiment has been properly performed and therefore represents a limiting factor. However it should be always considered that refolding after heat shock is a saturating event and therefore a proper time course analysis should be performed before starting any experiment.
Figure 1.
Overview of the experiment. In vivo refolding assay requires 3 to 4 days to be completed. On day 1 cells are seeded and transfected. The following day cells are splitted into a smaller format and on day 3 they are heat shocked. At this point is possible to perform the luciferase assay right after harvesting the cells or to save the cell pellet at -80°C and perform the assay in another moment
Figure 2.
Cell splitting. On day 2 cells are split into a 6-well plate. Each transfection will be diluted in a well in order to have all the transfection on the same plate. Each plate will correspond to a particular recovery time plus a non-shocked reference plate.
Figure 3.
Representative good result. A. Percentage of refolding directly measured by firefly luciferase activity. Each bar chart represents the percentage of refolding in absence (black bars) and in presence (red bars) of a molecular chaperone at different recovery time points. B. Correlation between time and refolding in absence (black line) and in presence (red line) of chaperone. The r squared values indicate good correlation.
In Fig.3A is shown a representative results where cells were collected 15, 30, 45, 60 and 120 minutes after heat shock in absence (black bars) and in presence (red bars) of molecular chaperone. Percentage of refolded luciferase increases with prolonged recovery time and with the transfection of the molecular chaperone. The correlation between refolding and time for the control (Fig. 3B, black line) and the molecular chaperone-transfected cells (Fig. 3B, red line) is shown.
Figure 4.
Representative bad result. A. Percentage of refolding directly measured by luciferase activity. Each bar chart represents the percentage of refolding in absence (black bars) and in presence (red bars) of a molecular chaperone at different recovery time points. B. Correlation between time and refolding in absence and in presence (red line) of chaperone. The r squared values indicate bad correlation.
Figure 4A. shows a representative result of an experiment not properly performed. Both the control (black bars) and the molecular chaperone-transfected cells (red bars) did not show an increase in protein refolding with the time. In addition, transfection of the molecular chaperone did not result in an increase of refolding. This poor correlation is confirmed in Fig. 4B showing an r squared value of 0.6619 and 0.1882 respectively for the control (black line) and the molecular chaperone-transfected cells (red line).
In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented. The whole assay can be performed in 3 to 4 days as shown by the overview in Fig. 1.
The robustness and linearity of the light signal produced by the firefly and the renilla luciferase represent a solid base for the reproducibility of the protocol.
The critical step of the assay is the choice of an efficient transfection reagent to ensure the overexpression of the molecular chaperone and the firefly luciferase. Reporter genes could be delivered also with other methods like adenoviral infection12 or electroporation. Another possibility is the use of a transgenic cell line where the expression of the chaperone is chemically inducible (e.g. a tetracycline-responsive promoter)13, while the gene of the firefly luciferase is stably transfected. This wide range of options makes thus possible the application of the assay to several cell lines, including primary cells.
Moreover, if a cell line is particularly sensitive to cycloheximide treatment, firefly luciferase protein translation could be arrested by the use a repressible/inducible system.
Since refolding activity can vary among the molecular chaperones, a titration of the amount of chaperone to be transfected should be always done. Once established in a particular cell line, the technique can be used to investigate how small molecules and/or gentic manipulations can affect chaperone activity. The assay can be performed also in a smaller format, like a 6- or even a 12-well plate.
One of the most attractive applications is the possibility to test entire libraries of compounds that can influence chaperone activity. In this particular case, cell lines stably transfected with the reporter gene as well as the chaperone are preferentially used, in order to minimize variations due to transfection efficiency.
This assay can be also used to investigate the role of co-activators or co-repressor of the molecular chaperones in protein refolding or how they influence refolding upon different stress stimuli (heat, oxidative stress, unfolded protein response).
Even if from a cell-based experiment is possible to have a more physiological readout of the effect of compounds and/or proteins on the activity of a particular chaperone, it is not possible to quantify the chaperone activity with the assay here presented. In this case would be more suitable a cell-free assay like in vitro refolding assay of firefly luciferase or β-galactosidase.
The authors have nothing to disclose.
Danilo Maddalo is a recipient of a research fellowship as Young Investigator Group (YIG) from the Karlsruhe Institute of Technology.
For this experiment a Luminometer form Perkin Elmer ‘1420 Luminescence Counter’ Vector Light′ was used
Programs for the Luminometer:
Renilla: Pump 1, 100 μl injection
Reaction Buffer: Pump1, 70 μl injection
Luciferin: Pump2, 30μl injection
For the buffers prepare these stock solutions in double distilled water:
GLY-GLY-buffer:
For 1 liter use 3,3g of Glycylglycin, 15ml of a 1M MgSO4 solution and 8ml of a 0,5M EGTA solution. Solve in water, adjust the pH to 7,8 and bring to a total volume of 1 liter.
Reaction buffer for Renilla/Coelenterazine buffer:
For 1 liter use 13,4ml of a 1M KH2PO4 solution, 86,6ml of a 1M K2HPO4 solution, 100ml of a 5M NaCl solution and 2ml of a 0,5M EDTA solution.
Company | Catalog/Product Number: | |
DMEM (Dulbecco’s Modified Eagle Medium 1X) | GIBCO | 41966029 |
Fetal Bovine Serum Gold | PAA | A15-151 |
X-treme Gene 9 DNA Transfection Reagent |
Roche | 06365809001 |
PBS Dulbecco’s Phosphate Buffered Saline 1X |
GIBCO | 14190-094 |
MOPS 3-(N-Morpholino)Propanesulfonic acid |
SIGMA-ALDRICH | M1254 |
Cycloheximide | SIGMA-ALDRICH | C7698 |
Passive Lysis Buffer 5X | Promega | E194A |
Glycylglycin |
SIGMA-ALDRICH Roth Roth |
G7278 3054.2 P027.1 |
KH2PO4 |
Roth Roth Roth Roth |
3904.1 6878.2 3957.1 8043.1 |
Luciferin Firefly | Biosynth | L8200 |
Coelenterazine | Biosynth | C7000 |
DTT (Dithiothreitol) | Roth | 6908.2 |
ATP (Adenosine Triphosphate) | Roche | 10519987001 |