Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways

Abstract

In certain physiological or pathological states (e.g., starvation, heat shock, or muscle atrophy) and upon drug treatments, the overall rate of protein degradation in cells may increase or decrease. These adaptations and pathological responses can occur through alterations in substrate flux through the ubiquitin-proteasome pathway (UPP), the autophagy-lysosomal system, or both. Therefore, it is important to precisely measure the activities of these degradation pathways in degrading cell proteins under different physiological states or upon treatment with drugs. In particular, proteasome inhibitors have become very important agents for treating multiple myeloma and very useful tools in basic research. To evaluate rigorously their efficacy and the cellular responses to other inhibitors, it is essential to know the degree of inhibition of protein breakdown. Unfortunately, commonly used assays of the activities of the UPP or autophagy rely on qualitative, indirect approaches that do not directly reflect the actual rates of protein degradation by these pathways. In this chapter, we describe isotopic pulse-chase methods to directly measure overall rates of protein degradation in cells by radiolabeling cell proteins and following their subsequent degradation to radioactive amino acids, which diffuse from cells into the medium and can be easily quantitated. While pulse-chase methods have often been used to follow degradation of specific proteins, the methods described here allow quantification of the total cellular activity in degrading either long-lived proteins (the great bulk of cell constituents) or the fraction with short half-lives. Moreover, by use of specific inhibitors of proteasomes or lysosomes, it is also possible to measure precisely the total contributions of the UPP or lysosomal proteases. These approaches have already been proven very useful in defining the effects of inhibitors, growth factors, nutrients, ubiquitination, and different proteasome activators on overall proteolysis and on substrate flux through the proteasomal and lysosomal pathways.

Keywords: 26S proteasomes; 3H-phenylalanine; Autophagy-lysosomal system; Liquid scintillation counting; Protein degradation; Pulse-chase; Radiolabeling; Ubiquitin; Ubiquitin-proteasome pathway (UPP).

Figure 1. Application of protein degradation assay protocols described in sections 3.1–3.3.

A. Protocol 3.1 measures the degradation of long-lived proteins (radiolabeled for 20 hours then chased for 2 hours to deplete short-lived radiolabeled proteins), whose degradation rate is constant. Therefore, precise degradation rate can be measured from the slope of the linear degradation curve. B. Application of Protocol 3.1: By measuring long-lived protein degradation in HEK293A cells treated with two mTOR inhibitors (Rapamycin or Torin1, which are anticipated to activate autophagy), we validated that the ability of cells to degrade protein was indeed elevated by these inhibitors. Figure adapted from [31]. C. Protocol 3.2 measures the degradation of mostly short-lived proteins labeled by a short pulse (5–20 min). Their mean degradation rate decreases rapidly over time due to the depletion of short-lived proteins, so that the degradation curve is non-linear, and it is not possible to calculate a constant degradation rate. D. Application of Protocol 3.2: The degradation of proteins pulse-labelled for 10 minutes was measured. Treatment with a Protein Kinase A (PKA) activator Forskolin greatly increased the amount of pulse-labelled proteins that were degraded over 1 hour. Note that the degradation curve is non-linear and no degradation rate could be calculated. The percentage of degraded proteins should be compared at each time points to draw conclusions. Figure adapted from [18]. E. Protocol 3.3 measures the degradation of long-lived proteins by a specific degradation pathway, by using a specific inhibitor to completely block this pathway. Subtracting these two degradation rates allow us to calculate the rate of inhibitor-sensitive degradation, which represents the ability of cells to degrade long-lived proteins through this particular pathway. F. Application of Protocol 3.3: To determine whether the increased degradation in cells treated with mTOR inhibitors (as in B) is caused by the lysosome pathway, the long-lived protein degradation rate was measured in cells whose lysosomes are completely inhibited by concanamycin A. This allow us to compare concanamycin A-sensitive (or lysosomal) degradation rates between control cells or cells treated mTOR inhibitor. We found using this approach, that lysosome contributes to the increased degradation in wild-type HEK293A or MEF cells, but not in cells with deficient autophagy (Atg5^−/−). Figure adapted from [31].

Figure 2. Calculation of the degradation rate of long-lived proteins

A. Raw Cpm counts for blank, TCA-soluble media samples, and cell lysate samples. B. To calculate how much ³H-Phenylalanine is released by protein degradation in each hour, multiply cpm increments by the volume of media during that hour. C. Combine the values from Fig 2B to calculate cumulative ³H-Phenylalanine released in 1–4 hours, which reflects the amount of degraded proteins (D0, D1, D2, D3, and D4). D. Calculate the ³H-Phenylalanine cpm remaining in proteins (R) and in total proteins (Total). E. Divide D0-D4 values by Total Cpm to calculate the percent degradation over 0–4 hours. Plotting these values (% cell protein degraded) will generate a linear curve, whose slope is the rate of protein degradation (% cell protein degraded / h).