We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.
Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.
Circadian rhythms refer to daily, predictable variations in biological function that are apparent throughout nature. They exist at every biological scale, from macroscopic behaviors like sleep-wake cycles, to molecular phenomena like the rhythmic abundance of biomolecules. In recent years, research into circadian rhythms has been transformed by the discovery of “clock genes” that are critical for circadian rhythm generation. Studies in clock gene knockout mice have revealed a central role for circadian rhythms in temporally organizing core cellular processes such as metabolism1. Among the ways circadian rhythms make this happen is by imparting a temporal structure to protein catabolism.
Several groups including ours have shown that the two major avenues for cellular protein catabolism, autophagy and the ubiquitin-proteasome system, are subject to diurnal rhythms2,3,4,5. Autophagy represents the lysosome-dependent arm of protein catabolism in which proteins of interest are delivered to this degradative organelle either through the construction of a novel vesicle (macroautophagy) or through direct translocation though a channel (chaperone mediated autophagy)6. The ubiquitin-proteasome system is the main non-lysosomal pathway, where proteins are poly-ubiquitinated and then fed into the proteasome, a macromolecular degradative machine found throughout the cytoplasm and nucleus7,8. Rhythms in autophagic and proteasomal activity are important because they likely play a role in cellular housekeeping. As a result, it is valuable to have a standardized procedure that can detect daily oscillations in protein catabolism that is compatible with pre-clinical disease models.
Here, we provide our protocol for quantifying diurnal variations in autophagic flux in mouse liver, which has served as the basis for work in our laboratory3,9. Our method is classified as a “turnover assay”10, an approach used by numerous groups to measure proteolytic activity (or flux). In this approach, protease inhibitors specific to lysosomes or proteasomes are administered to mice and then tissue samples are obtained after a fixed time interval. In parallel, tissue samples are obtained from mice subjected to sham injections. The tissue samples are homogenized and then biochemically separated to obtain the lysosome-enriched and cytoplasmic fractions. These fractions are then analyzed in parallel via western blotting using antibodies specific to macroautophagy markers (LC3b and p62) or proteasomal substrates (poly-ubiquitinated protein). Over time, animals injected with protease inhibitors accumulate proteins that would normally have been recycled. As a result, the rate of turnover is inferred by comparing the abundance of marker proteins in the protease-inhibitor treated samples to the sham-treated samples. By repeating this method at fixed time intervals across the day it is possible to reconstruct circadian variations in proteolysis (Figure 1A).
The protocol described here was approved by the Washington University in St. Louis Animal Care and Use Committee (IACUC).
1. Mouse Housing and Experimental Design
2. Preparation of Homogenization Solution, Inhibitor Stock Solutions, and Vials for Mouse Liver Collection
3. Protease Inhibitor Administration
4. Tissue Acquisition and Storage
5. Biochemical Fractionation of Liver Samples
NOTE: Figure 1B shows the fractionation scheme.
6. Western Blotting Readout
7. Data Analysis
NOTE: See Supplemental File “Sample Data”.
Representative data are presented in Figure 2A,B, and the quantification of these data are provided in Figure 2C,D (see also Supplemental File “Sample Data”). For simplicity, we have not depicted loading controls in Figure 2 but these should be obtained in parallel. Typically, western blots against β-actin are used for this purpose, but a total protein stain (such as Ponceau S) will suffice. The primary readout for autophagic flux at any given time point is the difference in the amount of macroautophagy specific markers (p62 or LC3b-II) between the leupeptin-treated versus sham samples in the lysosome enriched 3KP fraction divided by 2 (the number of hours between injection of protease inhibitor and tissue harvest). Similar data can be obtained from 20KP samples, which are also lysosome enriched, but our experience in mouse liver is that most of the signal segregates in the 3KP fraction. Typically, the results are normalized to the mean which simplifies comparison across independent experiments (Figure 2C,D). The primary readout for proteasomal turnover is the difference in the amount of Lys48-polyubiquitinated protein between the bortezomib-treated versus sham samples in the cytosolic (“Cyto”) fraction divided by 2. Because p62 can be a target of both macroautophagy and the proteasome3, an alternative marker for proteasomal flux is the change in p62 content +/- bortezomib in the 3KP fraction (see Sample Data). However, we find this marker is less robust compared to Lys48-polyubiquitinated protein and is best used as supportive data.
Figure 1: Experiment steps and sample processing. (A) Schematic of a typical time series experiment to measure daily rhythms in autophagic and proteasomal activity (flux) in mouse liver. (B) Fractionation scheme for obtaining lysosome-enriched and cytosolic liver protein fractions for western blot analysis. Please click here to view a larger version of this figure.
Figure 2: Sample experimental results of flux. Western blots from a representative time series experiment to measure daily rhythms in autophagic flux (A), and proteasomal flux (B) in mouse liver. Note that under leupeptin-treated conditions, p62 runs as ladder ranging from the monomeric form at about 50 kDa to SDS-stable complexes at about 250 kDa. Times of day are depicted in units of zeitgeber time (ZT), where ZT0 represents lights on and ZT12 represents lights off. Observation times that fall during lights off are shaded black. (C,D) Quantification of these data. Each data point represents the mean ± SE (n = 3). Statistical significance via one-way ANOVA is depicted. Please see the Supplemental File “Sample Data” for a tabular representation of these data. Please click here to view a larger version of this figure.
Supplementary file: Sample data. Laboratory analysis of densitometric data and subsequent statistical analysis. Please click here to download this file.
Our protocol describes a technically straightforward means of measuring biological rhythms in protein turnover in mice using commonly available molecular biology equipment. Because of the length of time series experiments and the number of biological samples involved, it is important to be consistent across the entire experiment regarding how the mice are injected, the timing of tissue acquisition and the biochemical processing of samples. The injection, euthanasia, and cervical dislocation steps may require operator practice prior to initiating a full-scale experiment. It is best for a single operator to perform all tissue dissections since different individuals dissect at different speeds, but if this is infeasible it may be worthwhile to increase the time between protease inhibitor injection and tissue harvest from 2 h to 3 or 4 h (provided this is done consistently across time points).
Common reasons for troubleshooting include variability in flux between biological replicate samples, and poor western blot quality. Regarding sample variability, this can be due to the use of multiple operators with different dissecting speeds or levels of experience. This can be mitigated by executing practice experiments until average dissecting times are less than 5 min per mouse. Alternatively, a single operator can be used to carry out dissections. High background on western blots can arise from uneven transfer, inadequate blocking, low quality primary antibody, or inadequate washing steps. Best results are achieved by overnight wet transfer of SDS-PAGE separated protein samples to PVDF in 10−20% methanol containing transfer buffer, using 10% non-fat dry milk for blocking, and extensive washing between steps (3x 10 min minimum on a mechanical rocker).
This technique has several limitations.First, the protocol described requires significant numbers of animals and is therefore resource intensive. While a minimal time series experiment spans 24 h, experiments of at least 2 cycles are ideal for confirming that the daily variations being observed in protein turnover are reproducible, and for estimating rhythm characteristics like periodicity and phase12. Because time must elapse between when the protease inhibitors are administered to mice and the time samples are harvested (to allow for targets of proteolysis to accumulate), the turnover measurements obtained represent an average activity over a 2 h interval rather than a point estimate. As a result, there is a limit to the resolution this assay can provide for detecting dynamic changes in protein catabolism. Finally, this protocol is optimized for mouse liver and the biochemical fractionation procedure presented here would likely need to be modified to efficiently obtain lysosomes from other tissue types.
This is the first method to directly measure daily rhythms in autophagic flux that also enables proteome-wide observations of this phenomenon. Future applications of this technique include analysis of proteolytic rhythms in various disease models, including autoimmune disease, cancer, and acute infection. In principal this method can be adapted to other mouse organs and to explanted human samples, which represents an exciting future application with high translational potential.
The authors have nothing to disclose.
This work was funded by RO1HL135846 and a Children’s Development Institute grant (PD-II-2016-529).
4x SDS PAGE Sample Buffer | Invitrogen | Cat# NP0008 | |
Bortezomib | EMD Millipore | Cat# 5.04314.0001; CAS: 179324-69-7 | |
Image Studio | LICOR | N/A | |
Immobilon-FL PVDF membrane 0.45 micron | Merck Millipore Ltd | Cat# IPFL00010 | |
K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb | Cell Signaling Technology | Cat#8081S; RRID:AB_10859893 | |
LC3a | Boston Biochem | Cat# UL-430 | |
LC3b antibody | Novus | Cat#NB100-2220; RRID:AB_10003146 | |
LC3b antibody | Cell Signaling Technology | Cat#2775; RRID:AB_915950 | |
Leupeptin | Sigma | Cat# L2884; CAS: 103476-89-7 | |
NuPAGE 4-12% Bis-Tris Midi Protein Gels | Thermo Fisher Scientific | Cat# WG1403BOX | |
NuPAGE LDS Sample Buffer (4x) | Thermo Fisher Scientific | Cat# NP0007 | |
P62-his | Novus | Cat# NBP1-44490 | |
Precision Plus Protein All Blue Prestained Protein Standards | Bio-Rad | Cat# 1610373 | |
Rabbit Anti-p62/SQSTM1 | Millipore-Sigma | Cat#P0067; RRID:AB_1841064 | |
rhPoly-Ub WT (2-7) (K48) | Boston Biochem | Cat# UC-230 | |
SDS-PAGE Midi-size Gels | Invitrogen | Cat# WG1403 | |
SIGMAFAST Protease Inhibitor Tablets | Millipore-Sigma | Cat# S8830 |