The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation
Figure 1
Effects of EGb 761 on basal proteasome activity. (a) HEK293 cells were treated for 24 h with indicated concentrations of EGb 761. Analysis of proteasomal peptidase (chymotrypsin-like) activity was assessed by the hydrolysis of SUC-LLVY-AMC in total cell lysates. Fluorescence of the cleaved AMC moiety was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated cells was arbitrarily set to 1; . (b–d) HEK293 cells with stable expressions of the proteasome reporter protein d2GFP (d2GFP-HEK) were treated for 24 h with indicated concentrations of EGb 761. Measurement of GFP fluorescence was used to investigate proteasomal degradation of d2GFP proteins. All achieved fluorescence intensities were finally adjusted to total protein content. (b) Cells were incubated with increasing concentrations of EGb 761 to investigate the specific, dose-depending effect on proteasome activity (a). Measurement of GFP fluorescence was used to assess the remaining d2GFP protein content as an indicator for an enhanced protein degradation by the proteasome. Values of vehicle-treated cells were arbitrarily set to 1. . (c) Previous assayed cells ((b); vehicle and 150 μg/mL EGb 761 treatments) were additionally incubated for 2 h with the proteasome inhibitor MG132 or DMSO as control. The addition of MG132 led to an increase of fluorescence intensities in control and EGb 761-treated cells. Inhibition of proteasome activity showed the specific modulation of GFP fluorescence through proteasomal d2GFP degradation. Values of vehicle-treated cells without MG132 were arbitrarily set to 1. . (d) Cells were treated for 24 h with 150 μg/mL EGb 761 or vehicle, followed by a chase with cycloheximide (CHX) to block synthesis of new d2GFP. Degradation kinetics of d2GFP was analyzed by measuring GFP fluorescence every 30 min. Fluorescence decay induced by CHX indicated the specificity of proteasomal d2GFP degradation. Values of each treatment at zero minutes were arbitrarily set to 1; . (e) HEK293 cells were treated for 2 h with 150 μg/mL EGb 761 or vehicle and RNA was extracted for qRT-PCR analysis. Relative expression ratio of proteasome genes PSMB5, PSMB6, and PSMB7 in EGb 761-treated cells to vehicle-treated cells is shown; . (a–e) All values are reported as mean ± S.D. and .
