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–Marie–Tooth 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.

中文翻译：

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解码 eIF2α 全磷酸酶和 PPP1R15A 抑制剂的选择性

蛋白质的可逆磷酸化控制着大多数细胞功能。与磷酸酶不同，蛋白激酶一直是流行的药物靶标，磷酸酶仍然是药物发现的挑战。Guanabenz 和 Sephin1 是磷酸酶调节亚基 PPP1R15A (R15A) 的选择性抑制剂，可延长 eIF2 α磷酸化的益处，从而保护细胞免受蛋白质抑制缺陷的影响。在小鼠中，Sehin1 可预防两种神经退行性疾病，即 Charcot-Marie-Tooth 1B (CMT-1B) 和 SOD1 介导的肌萎缩侧索硬化症 (ALS)。然而，R15A 抑制的分子基础尚不清楚。在这里，我们重组了人类重组 eIF2 α全磷酸酶，R15A-PP1 和 R15B-PP1，其活性取决于催化亚基 PP1（蛋白磷酸酶 1）和 R15A 或 R15B。该系统能够对这些全磷酸酶进行功能表征，并揭示 Guanabenz 和 Sephin1 诱导了 R15A 的选择性构象变化，通过对有限蛋白水解的抗性检测到。这改变了 eIF2 α的募集，防止其去磷酸化。这项工作表明磷酸酶的调节亚基是有效的药物靶点，并提供了将该概念扩展到其他磷酸酶的分子原理。