Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Our protocol allows measurement of the rate of autophagy and proteasomal activity in mouse tissues and is sensitive enough to detect circadian variations within these activities. This technique can be used in vivo to evaluate protein catabolism in cells and tissues and the required materials are relatively low-tech and accessible to any molecular biology lab. Demonstrating the procedure will be Mikhail Ryzhikov, a post-doc from my laboratory.

For fifteen minutes before each timepoint, warm the leupeptin and bortezomib stock solutions to room temperature and dilute the thawed bortezomib in sterile PBS to yield a 50 milligram per milliliter working solution. For protease inhibitor administration, intraperitoneally inject 40 milligrams per kilogram of leupeptin or 1.6 micrograms per kilogram of bortezomib into each experimental animal. For a negative control, inject mice with 0.5 milliliters of PBS.

Return each mouse to cages grouped by the type of treatment received as they are injected and record the time that the mice are treated or manipulated in a standardized table. Two hours after the injection, submerge the left liver lobe harvested from each sacrificed experimental animal in seven milliliters of ice cold homogenization buffer in appropriately labeled 15 milliliter conical tubes on ice. When all of the samples have been acquired, transfer the first sample and the entire volume of homogenization buffer into a 15 milliliter capacity Dounce homogenizer and homogenize the sample with 10 strokes of the loose piston and 15 strokes of the tight piston.

Return the resultant crude homogenate to its original tube and homogenize the next sample as just demonstrated. When all the samples have been homogenized, sediment the homogenate nuclei and debris by centrifugation and transfer the top four milliliters of each post-nuclear supernatant into new 15 milliliter tubes. Determine the protein concentration of this first supernatant fraction according to standard protocols and equalize the concentrations of all the samples to 2.1 milligrams per milliliter with fresh homogenization buffer as necessary.

For downstream analysis and quality improvement, aliquot 500 microliters of the normalized total protein fractions into 1.5 milliliter microcentrifuge tubes for minus 80 degrees Celsius storage. Transfer 1.5 milliliters of each of the remaining total protein fractions into individual microcentrifuge tubes and pellet the protein samples by centrifugation. Transfer one milliliter of the resulting second fraction supernatants into new microcentrifuge tubes on ice and aspirate the remaining supernatants.

Wash the 3K pellets two times in 1.5 milliliters of fresh cold homogenization buffer per wash, then resuspend the 3K pellet in 200 microliters of SDS-PAGE sample buffer and boil the samples at 95 degrees Celsius for five minutes. Spin the second fraction supernatants for 20 minutes at 20, 000 x g and four degrees Celsius and transfer the resultant third cytoplasmic fraction supernatants into a new microcentrifuge tube. For SDS-PAGE analysis, combine 150 microliters of the cytoplasmic fraction with 50 microliters of 4x SDS-PAGE sample buffer and boil the cytoplasmic fraction samples at 95 degrees Celsius for five minutes.

Aspirate the remaining supernatant from the 20K pellets and wash the samples two times with 1.5 milliliters of fresh cold homogenization buffer per wash. Then resuspend the 20K pellet in 100 microliters of SDS-PAGE sample buffer for boiling at 95 degrees Celsius for five minutes. For western blot analysis, serially dilute standard curve proteins one to three in 1x sample buffer for a total of six dilutions and load 10 microliters of each standard curve sample into one well of a 26 well four to 12%SDS-PAGE midi gel, then load a prestained commercial molecular weight standard.

To measure the autophagic flux, load 12 microliters of each 3K pellet sample into the appropriate wells. To measure the proteasomal flux, load 12 microliters of each cytoplasmic fraction into the appropriate wells. When all the control and experimental samples have been loaded, separate the protein samples by SDS-PAGE before transferring the proteins to polyvinylidene fluoride membranes according to standard protocols.

For analyzing macroautophagic flux, blot membranes containing the 3K pellet samples with anti-p62 or anti-LcB antibody overnight at four degrees Celsius. For analyzing proteasomal flux, blot membranes containing the cytoplasmic fraction samples with anti-lysine 48-Specific poly ubiquitin antibody overnight at four degrees Celsius. Then image the western blots using standard secondary antibodies and imaging devices.

To perform densitometric measurements on bands of interest for both the standard curve and experimental samples, open an appropriate image analysis software program and draw a long rectangle encompassing the protein monomer of interest at the bottom of the blot image extending to the top of the membrane. Copy and paste to move the rectangle to the remaining samples, keeping the rectangle consistent between samples. Save the quantification to a spreadsheet and generate a densitometric standard curve within the spreadsheet using the serially diluted standard sample in either linear or polynomial regression to obtain a best-fit standard curve equation.

Using this equation, extrapolate the quantity of the monomers of interest within the experimental samples. To obtain flux measurements, subtract the extrapolated protein quantity of each inhibitor treated sample from the average value of the PBS samples from the same timepoint. Then evaluate the statistical significance of the temporal variation in the protein turnover using one-way ANOVA.

The primary readout for autophagic flux at any given timepoint is the difference in the amount of macroautophagy specific markers between the leupeptin treated versus the sham samples in the lysosome-enriched 3K pellet fraction divided by two. Typically, the results are normalized to the mean which simplifies the comparison across independent experiments. Our procedure for measuring proteolytic flux depends on the ability of leupeptin or bortezomib to cause the accumulation of autophagy and proteasome substrates, which is a time-dependent process.

This technique has allowed exploration of how biological rhythms program liver protein catabolism. We hope it will pave the way for investigating the effects of disease on cellular housekeeping functions.