The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation
Figure 2
Modulation of proteasome activity by EGb 761 in cells expressing polyQ proteins. (a, c-d) HEK293 or (b) d2GFP-HEK cells were transiently transfected to express the polyQ fusion proteins with glutamine expansions of 25 (htt_Q25), 46 (htt_Q46), and 103 repeats (htt_Q103). Subsequent to settlement for 24 h cells were treated with 150 μg/mL EGb 761 or vehicle for different time periods and further investigated. (a) HEK293 cells expressing eGFP and polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle, followed by analysis of proteasomal peptidase activity in total cell lysates. Fluorescence of AMC moiety resulting from peptidase hydrolysis of SUC-LLVY-AMC was measured in the presence or absence of MG132 to achieve peptidase specificity. Values were adjusted to total protein content. Activity of vehicle-treated eGFP-expressing cells was arbitrarily set to 1; . (b) d2GFP-HEK cells expressing polyQ proteins (htt_Q25, Q46, and Q103) were treated for 24 h with EGb 761 or vehicle. Cell lysates of each sample were collected and analyzed by immunoblotting. Protein levels of d2GFP and proteasome subunit α1 were analyzed with their corresponding antibody to assess proteasome activity and status. Densitometric values of vehicle-treated and htt_Q25-expressing cells were arbitrarily set to 1; . (c-d) HEK293 cells expressing htt_Q25 and htt_Q103 for 24 h were analyzed by qRT-PCR for transcript levels of PSMB5, PSMB6, and PSMB7. (c) Quantification of basal expression ratio in cells expressing htt_Q103 to htt_Q25 (without EGb 761 treatment). (d) Analysis of cells additionally treated with EGb 761 or vehicle for 2 h. Expression ratio of proteasome genes in cells treated with EGb 761 compared to vehicle; . (a–d) All values are reported as mean ± S.D. and .
