Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

The degradation of misfolded proteins is crucial for maintaining protein homeostasis in cells.

To examine the degradation of misfolded proteins, take a culture plate containing transfected cells producing GFP-labeled, misfolding-prone proteins. Incubate the plate to facilitate the expression of the transfected protein until aggregates accumulate inside the cells.

Now, treat these cells with cycloheximide - a protein translation inhibitor - for different time intervals, and snap-freeze the cells. Cycloheximide inhibits new protein synthesis; this helps to study the time-dependent degradation of the pre-formed misfolded proteins.

Discard the spent medium, and treat the cells with lysis buffer containing a weak non-ionic detergent. Upon lysis, the detergent molecules solubilize the misfolded protein's monomers and oligomers, while the aggregates remain intact.

Centrifuge the contents to pelletize the aggregates, and discard the supernatant. Dispense the pellet into a sodium dodecyl sulfate, or SDS, buffer, and heat the sample. This treatment denatures the disordered aggregates, while the SDS-insoluble aggregates remain intact.

Load all the samples corresponding to different time intervals of cycloheximide treatment onto the SDS-PAGE gel. Due to their larger size, the SDS-insoluble aggregates remain intact in the well, while the monomers and SDS-soluble proteins migrate in the gel and form distinct bands.

Blot the samples and visualize using anti-GFP-antibodies. After longer cycloheximide treatment, the decreased intensity of the SDS-soluble proteins bands confirms misfolded protein degradation over time.

To carry out a degradation assay for Atxn1 82Q GFP, plate approximately 3 x 10⁵ 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. 4 to 5 hours after transfecting the cells with Atxn1 82Q-GFP/pRK5, under a fluorescence 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 3 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 2 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 4, 8, 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 microliters of ice-cold cell lysis buffer, and incubate on ice for 30 minutes.

Centrifuge the lysates in a benchtop centrifuge at 17,000 x g and 4 degrees Celsius for 15 minutes. Then, transfer the supernatant, which contains NP-40-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 NP-40-soluble fractions and NP-40-insoluble fractions resuspended from the pellets. Then, heat the samples at 95 degrees Celsius on a heat block for 5 minutes.

Add SDS-gel loading buffer to an aliquot of boiled NP-40-soluble and insoluble fractions. Load equal volumes of samples collected from all time points onto an SDS-PAGE gel. Detect NP-40-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-blot apparatus holding a 0.2-micron cellulose acetate membrane. Then, load 80 to 120 microliters of boiled NP-40-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.