The overall goal of this assay is to measure the cellular degradation rates of misfolded proteins. Misfolded proteins are associated with many neurodegenerative diseases. This assay helps to investigate the cellular protein quality control systems that are protectors against aberrant proteins.
Misfolding prone proteins may exist in different forms in cells. By combining cell fractionation with the cyclohexane method chase, the main advantage of this technique is to specifically measure the half life of the misfolded species. We use two model proteins as examples.
A highly aggregation prone polyglutamate protein, Atxn1 82Q and a conformationally unstable nuclear luciferase mutant. The protocol can also be applied to other misfolded protein measurements. To carry out a degradation assay for Atxn1 82Q GFP, plate approximately 3x10^5 HeLa cells in 35 millimeter plates with DMEM medium and 10%FBS.
Incubate the cells overnight so that they reach 40 to 60%confluence at the time of transfection. Four to five hours after transfecting the cells with Atxn1 82Q GFP PRK5, under a florescence microscope, use an excitation wavelength of 450 to 490 nanometers to examine the live cells for GFP expression in the nuclei, characterized by the presence of diffused as well as small speckles of GFP signals. Just before cycloheximide treatment, harvest one plate of cells by removing the medium and using three milliliters of ice cold PBS to wash the cells twice.
Then snap freeze the plate on dry ice. For the remaining plates of cells, remove the medium by vacuum aspiration and add two milliliters of fresh DMEM containing 50 micrograms per milliliter of cycloheximide. Also add 10 micromolar of the proteasome inhibitor, MG132 to one plate.
Incubate treated cells for four, eight, 12 and 16 hours before freezing on dry ice. After harvesting the MG132 treated cells at 16 hours, scrape the frozen cells from all the plates into 150 microliter of ice cold cell lysis buffer and incubate on ice for 30 minutes. Centrifuge the lysates in a benchtop centrifuge at 17, 000x g and four degrees Celsius for 15 minutes.
Then transfer the supernatant which contains MP40 soluble proteins to another tube. Rinse the pellets by gently adding approximately 200 microliters of 1x PBS to the tubes without disturbing the pellets. Carefully remove the PBS by aspiration or pipette, resuspend the pellets in 150 microliters of ice cold pellet buffer and then incubate them on ice for 15 to 30 minutes.
Next add 75 microliters of 3x boiling buffer into MP40 soluble fractions and MP40 insoluble fractions resuspended from the pellets. Then heat the samples at 95 degrees Celsius on a heat block for five minutes. Add SDS gel loading buffer to an aliquot of boiled MP40 soluble and insoluble fractions.
Load equal volumes of samples collected from all time points onto an SDS-PAGE gel. Detect MP40 soluble and SDS soluble Atxn1 82Q GFP by Western blot using anti GFP antibody and enhanced chemiluminescence. To examine SDS resistant Atxn1 82Q from the pellet fraction with a filter retardation assay, set up a dot plot apparatus holding a 0.2 micron cellulose acetate membrane.
Then load 80 to 120 microliters of boiled MP40 insoluble samples into each well of the dot blot apparatus. After filtering the samples through the membrane by vacuum, detect Atxn1 82Q GFP aggregates stuck on the membrane by anti GFP immunoblotting. It is critical to use a filter retardation assay to examine the levels of SDS resistant form over time.
This is because SDS soluble form may transform into SDS resistant form rather than being degraded. In this example, SDS resistant form is minimal and remains a similar level over the chase experiment. To carry out a degradation assay, after an overnight transfection of HeLa cells with NLS-luciferase-GFP, examine the live cells under an inverted fluorescent microscope for GFP expression with an excitation wavelength of 450 to 490 nanometers.
Just before cycloheximide treatment, harvest one plate of cells by removing the medium and use three milliliters of ice cold PBS to wash the cells twice. Then snap freeze the plate on dry ice. For the remaining plates, after removing the medium, add two milliliters of fresh DMEM containing 50 micrograms per milliliter of cycloheximide and add 10 micromolar of the proteasome inhibitor MG132 to one plate.
Treat the cells for 1.5, three, 4.5 and six hours before harvesting as just described, harvesting the MG132 treated cells at six hours. After harvesting the last time point of cells, scrape each plate into 150 microliters of ice cold cell lysis buffer and incubate on ice for 30 minutes. Incubate the samples on heat block at 95 degrees Celsius for five minutes, before carrying out SDS-PAGE and Western blotting according to the text protocol.
Add SDS gel loading buffer to whole cell lysates for a final concentration of 2%SDS and 50 millimolar DTT. After seeding and transfecting HeLa cells in 96 well plates according to the text protocol, 20 to 24 hours after transfection, examine the cells for GFP expression. Remove the medium, add approximately 200 microliters of 1x PBS to each well and then aspirate it to remove the residual DMEM.
Add 60 microliters of low fluorescence DMEM with 5%FBS and 50 micrograms per milliliter of cycloheximide and add 10 micromolar MG132 to one set of samples. With a fluorescence plate reader, measure the GFP signal, reading the plates every hour for up to eight to 10 hours. Export the data and carry out statistical analysis according to the text protocol.
In a steady state analysis, microscopically visible Atxn1 82Q GFP nuclear aggregates can be observed in 30 to 50%of HeLa cells 20 hours after transfection. As seen here by Western blot, the half life of Atxn1 82Q GFP in the SDS soluble fraction is around five hours. In contrast to a slight or no decrease in Atxn1 82Q GFP in the MP40 soluble fraction over 16 hours.
The degradation of Atxn1 82Q GFP is partially inhibited by treating cells with MG132. These images illustrate that 20 hours after transfection, NLS-luciferase DM mutant GFP aggregates become microscopically visible in five to 15%of HeLa cells. Western blots revealed that the half life of SDS soluble NLS-luciferase DM mutant GFP is two to three hours.
In addition, the florescence intensity of transfected cells also decreases over time upon cycloheximide treatment. At approximately six hours after cycloheximide treatment, soluble NLS-luciferase DM mutant GFP is largely degraded suggesting that the remaining fluorescence is generated from aggregated species resistant to degradation. These florescence images confirm that aggregated, but not diffused GFP, remains in cells nine hours after cycloheximide treatment.
Well the immunblotting based assay takes a few days to accomplish. The protein degradation rates can be obtained immediately after the cycloheximide chase using a fluorescence based assay. Both immunoblotting and fluorescence based assays can be applied to studies of other misfolded protein.
The latter is also suitable for high throughput screening for identifying macromolecules or small compounds that can modulate the degradation of misfolded proteins. It is important to remember that for some misfolded proteins such Atxn1 82Q, the half life can only be measured through cell fractionation and immunoblotting and this is because the misfolded species that degraded quickly are only a small portion of the overall Atxn1 82Q expression. To uncover the mechanism of a protein quality controls, protein degradation assays need to be coupled with analysis of the steady state levels of aggregates as well as that they are partitions in different cell fractions.
Other experiments like in vitro protein folding and aggregation assays can also be performed. After watching this video, you should have a good understanding of how to measure the degradation rates of misfolding proteins using different strategies. Thanks for watching and good luck with your experiments.
