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First published online 3 April 2007
doi: 10.1242/jcs.03438

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Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease

Paula van Tijn^1,*, Femke M. S. de Vrij^1,*,, Karianne G. Schuurman¹, Nico P. Dantuma², David F. Fischer^1,, Fred W. van Leeuwen^1,¶ and Elly M. Hol^1,**

¹ Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands
² Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, Box 285, SE-17177 Stockholm, Sweden

View larger version (82K): [in this window] [in a new window]   Fig. 1. UBB+1 is degraded by the UPS at low expression levels. (A,B) Western blot of cell lysates of HeLa cells transiently transfected with inducible Tet-off CMV-UBB+1 vectors treated with decreasing Dox concentrations. UBB+1 was detected with anti-UBB+1 antibody (Ub3) (De Vrij et al., 2001) in cells without proteasome inhibitor treatment (A) or in cells treated overnight with 1 µM proteasome inhibitor MG132 (B). CMV-UBB+1 pcDNA3 (UBB+1) and empty pcDNA3 vector (control) transfections served as controls. Arrows in B indicate additional UBB+1 expression after proteasome inhibitor treatment. Protein input was equal in all lanes as determined by Bradford protein measurement (not shown); this is a representative experiment of two duplicate experiments. Molecular mass in kDa is indicated on the left. *UBB+1; **Ub-UBB+1.

View larger version (60K): [in this window] [in a new window]   Fig. 2. High levels of UBB+1 inhibit the proteasome. Flow cytometric analysis of UbG76V-GFP HeLa cells for GFP fluorescence as an indication of UPS inhibition (% of cells that are GFP positive). (A) Cells were transiently transfected with empty pcDNA3 vector (control), CMV-Ub wild-type pcDNA3 (Ubwt), CMV-UBB+1K29,48R pcDNA3 (+1K29,48R) or CMV-UBB+1 pcDNA3 (UBB+1) or treated with 100 nM epoxomicin (epox) or 1 µM MG132. Significant accumulation of GFP compared with empty vector control is marked with an asterisk (*P<0.005, ANOVA Bonferroni). (B) UbG76V-GFP cells were transiently transfected with the inducible Tet-off UBB+1 expression system and treated with decreasing concentrations of Dox. Significant increase in the percentage of GFP-positive cells compared with levels in cells treated with 1000 ng/ml Dox is marked by an asterisk (*P<0.05, ANOVA Bonferroni). Results are the means ± s.e.m. of three or four independent duplicate experiments. (C) Representative flow cytometric scatter plots of UBB+1 Tet-off transfected cells treated with 1000, 0.01, 0.0001 and 0 ng/ml Dox as shown in B. GFP-positive cells were detected in the region set as R1. The mean GFP fluorescence and the percentage of cells that are GFP-positive are indicated on the right.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Accumulation of UBB+1 is reversible after shutting off expression. Flow cytometric analysis of the percentage of UBB+1 (A) or UBB+1/GFP (B) positive cells at 0 hours, 12 hours and 36 hours after shutting down maximal UBB+1 expression. UbG76V-GFP HeLa cells were not transfected (control), transfected with empty pcDNA3 vector (e) or transfected with the UBB+1 Tet-off vectors. After 64 hours of maximal expression (absence of Dox) a baseline sample was taken (0 hours). UBB+1 expression was continued at maximal levels (Dox–) or shut down by addition of 10 ng/ml Dox (Dox+) and samples were analyzed after 12 hours and 36 hours of Dox treatment. Results are the means ± s.e.m. of two or three independent duplicate experiments, *P<0.01; #p=0.099 ANOVA between groups indicated at 36 hours.

View larger version (148K): [in this window] [in a new window]   Fig. 4. Lentiviral transduction targets a heterogeneous cell population in cortex slice cultures. GFAP (red) and NeuN (blue) double staining on LV-Ub-M-GFP transduced organotypic cortex slice cultures of C57Bl/6 mice revealed mostly GFAP-labelled GFP-positive glia, but also GFP-positive neurons. Arrows indicate transduced neurons, positive for both GFP and Neu-N. Bar, 50 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. UBB+1 is degraded by the proteasome in cortex slice cultures. Organotypic cortex slices of C57Bl/6 mice were transduced with LV-UbG76V-GFP, LV-UBB+1 or LV-UBB+1K29,48R. Both the UPS reporter protein UbG76V-GFP (green) and UBB+1 (red) are efficiently degraded by the 26S proteasome and only accumulate after treatment with proteasome inhibitor. The lysine mutant of UBB+1, UBB+1K29,48R, is not degraded by the proteasome and accumulates without inhibitor treatment. – epox, not treated with epoxomicin; + epox, treated overnight with 1 µM epoxomicin. Bar, 100 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 6. UBB+1 remains present after washout of inhibitor in cortex slice cultures. Overnight incubation of cortex cultures transduced with LV-UbG76V-GFP or LV-UBB+1 with the reversible proteasome inhibitor MG132 (10 µM) resulted in accumulation of both proteins. Washing out the reversible inhibitor reactivated the proteasome, as shown by the degradation of the proteasome reporter substrate UbG76V-GFP. However, UBB+1 remained in a considerable number of cells after reactivation of the proteasome. Transduction with the LV-UBB+1K29,48R control construct gave rise to accumulation of the UBB+1 protein regardless of proteasome inhibitor treatment. UbG76V-GFP is depicted in green, UBB+1 in red, and the nuclear staining (TO-PRO) in blue. Bar, 500 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 7. The UPS reporter system in cortex cultures of UbG76V-GFP transgenic mice. (A) UbG76V-GFP tg organotypic cortex cultures without treatment with proteasome inhibitors. (B) UbG76V-GFP tg cortex cultures treated with 1 µM epoxomicin. The GFP-reporter substrate only accumulated after proteasome inhibition. Bars, 50 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 8. High Ad-UBB+1 expression causes proteasome inhibition in cortex cultures. High levels of UBB+1 expression with Ad-UBB+1 led to accumulation of UBB+1 without inhibitor treatment. (A) Representative western blot of HEK293 cell lysates transduced with equal MOI of LV-UBB+1 (left lane) or Ad-UBB+1 (right lane). Equal amounts of protein were loaded per lane, as confirmed by Coomassie Blue staining of total protein load of the same lanes shown on the right. The blot was stained with anti-UBB+1 antibody Ub3 and quantified with Imagepro software (quantification not shown). (B,C) Organotypic cortex slice cultures of UbG76V-GFP tg mice were transduced with LV-UBB+1, which did not induce UBB+1 accumulation (B) or Ad-UBB+1, which did result in many UBB+1-immunopositive cells (C). (D):UBB+1 accumulation after adenoviral transduction led to accumulation of UbG76V-GFP (arrows). Bars, 250 µm (B); 500 µm (C); 50 µm (D).

View larger version (32K): [in this window] [in a new window]   Fig. 9. UBB+1 properties shift from UPS substrate to inhibitor. (1) UBB+1 mRNA and translation levels are constant throughout life (Fischer et al., 2003; Gerez et al., 2005). In non-diseased tissue, the 26S proteasome is capable of degrading all the translated UBB+1 and accumulation of UBB+1 is not present. (2) Owing to various causes such as disease or aging, the efficiency of proteasomal degradation can decrease, leading to a diminished degradation of UBB+1. (3) The levels of translated UBB+1 exceed the degradation capacity of the proteasome and surpass the accumulation threshold. Accumulated UBB+1 now holds UPS inhibitory properties itself, which can aggravate the initial decrease in UPS activity.