Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

Abstract

The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot-MarieTooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A-PP1 and R15B-PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

“…Sal003 inhibits dephosphorylation of eIF2α-P by targeting the interaction of PP1 with either CReP or GADD34 regulatory subunits (Hetz et al, 2013). To determine which subunit is important for SNAT2 expression, we tested the effects of Guanabenz (Guan) and Sephin 1 (Seph), which target GADD34/PP1 but not CReP/PP1 (Carrara et al, 2017; Das et al, 2015; Hetz et al, 2015; Tsaytler et al, 2011). These inhibitors have similar structures; however, Guanabenz is also a potent agonist of the α2-adrenergic receptor (Das et al, 2015; Tsaytler et al, 2011).…”

“…3E), with Sal003 having a more prominent effect. Sephin 1 is a very selective GADD34/PP1 inhibitor (Carrara et al, 2017; Das et al, 2015); in contrast, Sal003 has an additional inhibitory effect on PP1 when, instead of GADD34, it contains the constitutively-expressed regulatory subunit CReP (Choy et al, 2015). We next tested if the effects of GADD34/PP1 on Golgi fragmentation involve the well-known eIF2α-P-mediated responses in ISR.…”

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1‐Disrupting Peptides

“…Protein phosphatase‐1 (PP1) is a Ser/Thr‐specific phosphoprotein phosphatase (PPP) that has central roles in cellular and organismal physiology, and has been implicated in several human diseases associated with deregulated phosphorylation . PP1 holoenzymes each contain a conserved catalytic subunit with an active‐site pocket that is highly similar to that of other PPPs, and one or two regulatory proteins that determine substrate specificity, cellular localization, or activity .…”

Guanabenz treatment improves Oculopharyngeal muscular dystrophy phenotype

“…The third pathway is operated by the activating transcription factor 6 (ATF6), a receptor that under ER stress and its dissociation to Grp78 is translocated to the Golgi where it is cleaved to produce a fragment which then acts as a transcription factor to promote expression of XBP1 mRNA and other proteins involved in protein folding. It has been proposed that GA binds to PPP1R15A, thus preventing its binding to PP1c (37,38) and prolonging eIF2α phosphorylation with the effect of reducing the accumulation of misfolded proteins in the ER (37,39), but these data are controversial (40,41).…”