Background

Regulation of protein abundance is crucial for the maintenance of normal cell function; all proteins in the cell are constantly being degraded and replaced. Specifically, rapid protein degradation plays an important role in regulating protein abundance in a variety of cellular processes, including cell cycle, signal transduction, and differentiation (Goldberg, 2003). Studies on protein degradation rate and half-life are important for understanding protein function, developing drugs to regulate the degradation of disease-specific proteins, and researching small molecule compounds to regulate protein levels (Paiva and Crews, 2019). There are two main protein degradation pathways in eukaryotic cells—the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. The former is the main pathway of protein degradation in cells and is involved in the degradation of various proteins (Glickman and Ciechanover, 2002). It degrades proteins in cells in a specific, highly complex mechanism, and can be reversed by deubiquitinating enzymes (Sun et al., 2020). Autophagy-lysosome pathway is a self-degradation process, which degrades proteins through lysosomal mediation, by coating them and forming an autophagic lysosome, to fulfill the metabolic needs of the cells. Both pathways play vital roles in maintaining the normal protein metabolism of cells, and abnormal protein degradation may lead to many diseases, especially neurodegenerative disorders and cancer (Huang et al., 2018; Park et al., 2020).

The half-life of proteins varies widely, from a few minutes to days, and different rates of protein degradation are important for protein function. The classical method for protein degradation detection is the pulse-chase assay (Fritzsche and Springer, 2014); this assay is based on the addition of radionuclide-labeled amino acids, such as 35S-labeled methionine, to cell culture medium. The change process of a specific protein in the cells can be detected by immunoblotting or other methods. However, this method uses isotopes, which have some negative effects on the health and safety of researchers, and the operation process is complicated. Another common approach to study protein degradation is the protein translation blocker cycloheximide (CHX), which we will describe in detail later. In recent years, mass spectrometry technology has developed to detect the degradation of the primary structure of proteins. Therefore, protein degradation detection by mass spectrometry can be used to study protein structure, function, and quality control (Spradlin et al., 2021). In addition, it has been reported that CuAAC (copper-catalyzed azide–alkyne cycloadditions) can be used to label newly synthesized proteins in cells and to study protein degradation (Lu et al., 2013). Besides, protein degradation can be studied through the addition of pharmacological inhibitors of the degradation pathway to cells (Hanzl and Winter, 2020). For example, proteasome inhibitors such as MG-132, epoxomicin, and bortezomib block protein degradation via the ubiquitin-proteasome pathway. Inhibitors such as chloroquine and bafilomycin A1 work by neutralizing the acidic pH in the lysosome, which is necessary for the function of lysosomal protease.

Here, we describe a method that inhibits protein synthesis by adding CHX to assess the degradation of the target protein over time. CHX is a global translational inhibitor that can block the eukaryotic ribosome translocation process (Schneider-Poetsch et al., 2010). CHX is a product of Streptomyces griseus that can be used to determine the half-life of proteins or enzymes in molecular biology. It is noteworthy that CHX can inhibit eukaryotic protein synthesis but not prokaryotic protein synthesis. The advantages of CHX are that it is easy to operate, does not require considering the effect of ongoing protein synthesis, and allows for high-throughput screening. On the other hand, its main disadvantages are i) blocking all protein translation without specificity, and ii) having high cytotoxicity, able to cause large-scale cell death after prolonged treatment, so it is not suitable for proteins with a slow degradation rate. Therefore, CHX needs to be selected according to the appropriate conditions.

CHX chase assay visualizes protein degradation kinetics through CHX treatment over different times and western blotting analysis. Briefly, cells are treated with CHX at a suitable concentration, and then harvested at different timepoints. Next, western blot is employed to analyze the expression levels of a given protein. A short half-life protein will decrease in abundance over time, while a long half-life protein could exhibit little change in abundance. Thus, the CHX chase assay visualizes protein degradation efficiently without the use of radioactive isotopes, which provides an easy and reliable means to observe the change of intracellular protein levels and therefore could contribute to the study of therapeutic targets for a range of diseases.