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. 2009 Feb 26;4(2):e4597. doi: 10.1371/journal.pone.0004597

Figure 8. Proteasome function is impaired by mutant or loss of PINK1.

Figure 8

Proteasome activity was measured from SH-SY5Y cells expressing mutant PINK1 (A), and PC12 cells expressing siRNA against PINK1 (B, C, D). A) Fluorescence of fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) is positively correlated with proteasome function. No statistically significant changes were detected in proteasome activity between control SH-SY5Y cells and the cells expressing wild type PINK1 (n = 8, p = 0.484, paired student t test). There was a statistically significant decrease of proteasome activity in the SH-SY5Y cells expressing L347P-PINK1 (23% reduction, n = 7, p = 0.018, paired student t test) or in SH-SY5Y cells expressing E417G-PINK1 (19.4% reduction, n = 8, p = 0.012, paired student t test) compared to cells expressing wild type PINK1. MG132, a proteasome inhibitor, was used as a negative control. The bottom panel is a Western analysis of the above samples with the 20S α subunit Ab for normalization. B) Proteasome activity was measured in 20 µg of cell lysate isolated from wild type control PC12 cells (open diamond) or SiPINK1-4 PC12 cell line (filled circle) for 60 min after 30 min incubation. Wild type PC12 cells lysate treated with MG132 (filled triangle) was used as a negative control. The result revealed that the kinetic of proteasome activity monitored over 60 min was markedly decreased in the cells with reduced PINK1. The bottom panel is a Western analysis of the above samples with the 20S α subunit antibody for normalization. C) Histographic presentation for Figure 7B. The reduction of PINK1 by siRNA impairs the proteasome activity (31.8% reduction, n = 8, p = 0.01, ANOVA). Experiments were repeated with SiPINK1-2 PC12 cell line, and consistent results were obtained (data not shown). D) PINK1 mediated proteasome activity deficit confirmed by another independent method in the HeLa cells. Compared to control (CFP-de transfection), siRNA against PINK1 (siPINK1) knocked down PINK1 and led to a sigfinicant inhibition of CFP degradation (p = 0.0001, ANOVA) to an extent similar to direct proteasome inhibition by MG132 (p = 0.0014, ANOVA). A scrambled siRNA (siSCR) had no effect (p = 0.876, ANOVA). The RNAi sequences are: GAGAGGUCCAAGCAACUA TT and CCUGGUCGACUACCCUGAU TT.