Establishing ocular cell lines to monitor the ubiquitin-proteasome system (UPS) activity in cells with fluorescent GFP reporters.
- Background: The Ubiquitin-proteasome system (UPS) plays a critical role in nearly every biological process that includes the removal of undesired damaged proteins. Reduction of UPS capacity is associated with aging and several age-related diseases. In order to understand the relationship between the accumulation of damaged proteins and aging or age-related diseases a reliable experimental system ... read moreis necessary for measuring capacity of UPS. We establish an experimental procedure to measure capacity of UPS based on a GFP-fusion UPS reporter, Ub-G76V-GFP, constitutively expressed in Hela cells, a cell line prepared by other groups (Dantuma et al., 2000). We develop a method for cyclohexamide chase assay of the UPS reporter by utilizing fluorescence spectroscopy. Methods and Findings: Cyclohexamide (CHX) chase assay of Ub-G76V-GFP was performed to measure the degradation kinetics of the UPS reporter. CHX chase assay was chosen as a method in order to eliminate the concern about the potential changes in the transcriptional rate of the UPS reporter. We used fluorescence spectrometry (fluorescence plate reader) and Western blotting to measure the level of Ub-G76V-GFP. The Hela cells constitutively expressing Ub-G76V-GFP were first incubated for 3 hours with a proteasome inhibitor, MG132, in order to accumulate the reporter to an observable level. After MG132 was removed, cells were incubated with or without cyclohexamide or cyclohexamide and MG132 combined. These cells were harvested at 0, 2, 4 and 6 hours during incubation and lysed in a buffer containing detergent (1% Triton X-100 and 0.03% SDS), and the level of Ub-G76V-GFP in the whole cell lysate and detergent soluble fraction was measured. With cyclohexamide alone, most Ub-G76V-GFP was degraded within 4 hours. Without cyclohexamide, most of Ub-G76V-GFP was still degraded in 6 hours. As expected, MG132 significantly blocked the degradation of Ub-G76V-GFP, although some of the Ub-G76V-GFP (~40%) was still degraded over 6 hours. Essentially, Ub-G76V-GFP in the whole cell lysate and detergent soluble fraction showed the same degradation rate, and almost no Ub-G76V-GFP was detected in the detergent insoluble fraction. Conclusions: In this project, a method of monitoring the UPS reporter was established. This procedure can be used in the future to examine the effect of aging and protein -damaging stress on the capacity of the UPS. When using this method, we recommend performing the experiment within 4 hours because of the rapid degradation kinetics of Ub-G76V-GFP.read less
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