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Published on: May 1, 2020
PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz
Ana Crespillo-Casado1, Joseph E Chambers1, Peter M Fischer2,3
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
Abstract:
Dephosphorylation of translation initiation factor 2 (eIF2α) terminates signalling in the mammalian integrated stress response (ISR) and has emerged as a promising target for modifying the course of protein misfolding diseases. The [(o-chlorobenzylidene)amino]guanidines (Guanabenz and Sephin1) have been proposed to exert protective effects against misfolding by interfering with eIF2α-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex. Surprisingly, they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation. Furthermore, eIF2α-P dephosphorylation, assessed by a kinase shut-off experiment, progressed normally in Sephin1-treated cells. Consistent with its role in defending proteostasis, Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response. However, repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2α (eIF2αS51A). These findings challenge the view that [(o-chlorobenzylidene)amino]guanidines restore proteostasis by interfering with eIF2α-P dephosphorylation.
Insights
[(o-chlorobenzylidene)amino]guanidines like Sephin1 do not restore proteostasis by inhibiting eIF2α-P dephosphorylation. In vitro and cellular studies show these compounds are inert in disrupting the PP1-PPP1R15A complex, challenging their proposed mechanism.
Area of Science:
- Molecular Biology
- Cellular Stress Response
- Neurodegenerative Diseases
Background:
- The integrated stress response (ISR) involves dephosphorylation of translation initiation factor 2 (eIF2α) to terminate signaling.
- Protein misfolding diseases are targeted by modulating eIF2α-P dephosphorylation.
- [(o-chlorobenzylidene)amino]guanidines (Guanabenz, Sephin1) were proposed to protect against misfolding by disrupting the PP1-PPP1R15A complex, inhibiting eIF2α-P dephosphorylation.
Purpose of the Study:
- To investigate the mechanism of action of [(o-chlorobenzylidene)amino]guanidines, specifically Sephin1, in the context of protein misfolding diseases.
- To determine if Sephin1 interferes with eIF2α-P dephosphorylation in vitro and in cellular models.
- To evaluate the role of eIF2α-P dephosphorylation in Sephin1's proposed proteostasis-restoring effects.
Main Methods:
- In vitro assays to assess the stability of the PP1-PPP1R15A holophosphatase complex and substrate-specific dephosphorylation.
- Kinase shut-off experiments in cells to monitor eIF2α-P dephosphorylation kinetics in Sephin1-treated cells.
- Analysis of Sephin1's effects in wildtype, Ppp1r15a-deleted, and Eif2s1 (eIF2αS51A) CRISPR-edited cells.
Main Results:
- Sephin1 and Guanabenz were found to be inert in vitro, showing no effect on PP1-PPP1R15A complex stability or eIF2α-P dephosphorylation.
- eIF2α-P dephosphorylation proceeded normally in Sephin1-treated cells, even in the absence of PPP1R15A or a phosphorylatable eIF2α.
- Sephin1 attenuated the IRE1 branch of the unfolded protein response, but this effect was observed in ISR-deficient cells, suggesting an alternative mechanism.
Conclusions:
- The proposed mechanism of [(o-chlorobenzylidene)amino]guanidines restoring proteostasis by inhibiting eIF2α-P dephosphorylation is challenged.
- Sephin1's protective effects, if any, likely operate through pathways independent of direct interference with the PP1-PPP1R15A complex or eIF2α-P dephosphorylation.
- Further research is needed to elucidate the true mechanism underlying the cellular effects of these compounds.
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