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Science 332 (6025): 91-94

Copyright © 2011 by the American Association for the Advancement of Science

Selective Inhibition of a Regulatory Subunit of Protein Phosphatase 1 Restores Proteostasis

Pavel Tsaytler1, Heather P. Harding2, David Ron2, and Anne Bertolotti1,*

1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
2 Institute of Metabolic Sciences, University of Cambridge, Cambridge CB2 0QQ, UK.


Figure 1
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Fig. 1. Guanabenz protects cells from deleterious accumulation of misfolded proteins in the endoplasmic reticulum. (A) Assessment of the number of viable, blasticidin-resistant Min6 cells, after transduction with blasticidin resistance (blst)–tagged lentiviruses encoding a misfolding-prone fusion of insulinAkita with green fluorescent protein (GFP) (10), treated with the indicated concentrations of guanabenz. Values, corresponding to the cells’ ability to reduce WST-8 into formazan, were normalized to those of cells transduced with blst-tagged lentiviruses encoding cytoplasmic GFP. (B) Same as (A) with INS-1 cells. (C) Viability of HeLa cells assessed by the reduction of WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] into formazan, after treatments with tunicamycin (2.5 μg/ml; Tm) for 12 or 24 hours, with or without the indicated concentrations of guanabenz. (D) Dose-dependent protection of HeLa cells by guanabenz, from ER stress induced by 6 hours of exposure to tunicamycin. Data are means ± SD (n = 4). **P ≤ 0.001; ***P ≤ 0.0001.

 

Figure 2
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Fig. 2. Guanabenz increases chaperone availability by attenuating translation recovery after ER stress. (A) Immunoblot of the indicated proteins in lysates of HeLa cells treated with guanabenz (50 μM) or tunicamycin (2.5 μg/ml; Tm) for the indicated times. (B) Autoradiogram of [35S]methionine-labeled proteins in cell lysates resolved by NuPage after a 10-min labeling pulse of HeLa cells exposed to guanabenz (50 μM) or Tm (2.5 μg/ml) for the indicated times. Lower panel is a photomicrograph of the Coomassie-stained gel. (C) Same as (B) except that cells were treated with Tm in the presence or absence of guanabenz. (D) Immunoblots of HeLa cell lysates from cells treated with tunicamycin (2.5 μg/ml) in the presence or absence of guanabenz (50 μM) for the indicated times. (E) Immunoblots of BiP recovered in SDS-resistant high–molecular weight (HMW) complexes (12) or in the total cell lysates from HeLa cells left untreated or treated with tunicamycin in the presence or absence of guanabenz. Representative results of three independent experiments are shown.

 

Figure 3
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Fig. 3. Guanabenz selectively binds PPP1R15A and disrupts the PPP1R15A-PP1c complex. (A) Immunoblots of PPP1R15A immunoprecipitates (IP) recovered from lysates of HeLa cells treated with tunicamycin for 10 hours, in the presence of guanabenz for the last 5 hours where indicated (50 μM). SN, supernatant of the immunopurification. (B) FLAG immunoprecipitates of HeLa cells overexpressing FLAG-tagged PPP1R15A or PPP1R15B, untreated or treated with 50 μM guanabenz for 6 hours, analyzed by immunoblotting. (C) FLAG-PPP1R15A immunoprecipitates from unstressed HeLa cells, washed with indicated concentrations of guanabenz and analyzed by immunoblotting. (D) Immunoblots showing that PPP1R15A but not PPP1R15B, expressed in rabbit reticulocyte lysate (input), selectively bound to biotinylated guanabenz immobilized on neutravidin beads. (E) In vitro translated PPP1R15A bound biotinylated guanabenz immobilized on neutravidin beads eluted with 1 mM guanabenz. Immunoblots show inputs, eluates, and beads before and after elution. Note that PP1c did not bind to the biotinylated guanabenz. (F) Top: Schematics of human PPP1R15A. Lower left: Same as (D) except that proteins were in vitro translated in the presence of [35S]methionine and revealed by phosphorimaging. Lower right: Coomassie-stained gel showing bacterially expressed and purified PPP1R15A230–674 fragment (input) selectively retained on neutravidin beads in the presence of biotinylated guanabenz (bound). Representative results of at least three independent experiments are shown in each panel.

 

Figure 4
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Fig. 4. The ER stress–cytoprotective activity of guanabenz is mediated by inhibition of PPP1R15A. (A) Viability of wild-type MEFs or mutant cells (mut/mut) lacking PPP1R15A or PPP1R15B activity after exposure to the indicated concentrations of guanabenz for 48 hours. Data are means ± SD (n = 4). ***P ≤ 0.0001; n.s., not significant. (B) Viability of wild-type or Ppp1r15a mutant (mut/mut) MEFs exposed to tunicamycin (1 μg/ml; Tm) for 6 hours with the indicated concentrations of guanabenz, assessed by the ability to reduce WST-8 into formazan. The reducing activity of cells of either genotype exposed to tunicamycin without guanabenz was normalized to 1.

 


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