In a previous study by our group, we showed that an activator of the PERK pathway was protective in chemical- and viral-induced tauopathy cell models and in vivo. Another study revealed that indirect activation of the PERK pathway by inhibition of its repressors, led to lower levels of tau accumulation. Studies with cells carrying PERK risk alleles from patients with tauopathy concluded that enhanced PERK signaling can decrease vulnerability to ER stress-associated damage. So far, several in vitro and in vivo studies have demonstrated that modulation of the PERK pathway is a promising therapeutic approach in tauopathies. Furthermore, PERK activation is known to increase cell survival, particularly via the NRF2-pathway, which induces the transcription of antioxidative genes, helping cells to cope with UPR-induced oxidative stress. Phosphorylation of eIF2α reduces the flux of protein in the ER by causing translation arrest, alleviating ER protein stress. Activation of the PERK pathway ultimately leads to the phosphorylation of its downstream effectors-eIF2α and NRF2. More recently, it was shown that the PERK luminal domain in a transient tetrameric state appears to be crucial for an efficient signal transduction. The first oligomeric structures described were interdimers, proposed to contribute to PERK trans-autophosphorylation at residue Thr980, consequently stabilizing the PERK activation loop, a structural component which is important for the interaction of PERK and its natural substrate proteins, e.g., eIF2α. Although the exact mechanism for PERK activation is still not fully understood, accumulating evidence suggests that PERK is activated upon conformational change and oligomerization. Accumulation of unfolded or misfolded proteins or other factors, e.g., changes in calcium homeostasis, trigger ER stress and lead to BiP dissociation and activation of the PERK pathway. Under non-stressed conditions, PERK is kept at an inactive state by the chaperone BiP (binding immunoglobulin protein). PERK is an ER (endoplasmic reticulum) transmembrane protein with an ER lumen regulatory domain and a cytoplasmic serine/threonine kinase domain. In recent years, there has been growing evidences that activation of the PERK (pancreatic endoplasmic reticulum kinase) pathway, a major branch of the UPR (unfolded protein response) involved in restoring protein homeostasis, may be a promising therapeutic target in the context of tauopathies. Tauopathies are a heterogeneous group of neurodegenerative disorders characterized by an abnormal phosphorylation and aggregation of the microtubule-associated protein tau with a progressive accumulation of tau aggregates in neurons. These activators will be useful for deepening our understanding of the PERK signaling pathway, and may also lead to the identification of new therapeutic drug candidates for neurodegenerative tauopathies. Overall, our data show that a cell-free luciferase-based assay with the recombinant human PERK kinase domain and SMAD3 as substrate protein is capable of detecting PERK activation, which enables to screen large compound libraries for direct PERK activators, in a high-throughput-based approach. Finally, we confirmed the applicability of the assay by measuring PERK activation by MK-28, a recently described PERK activator. Additionally, our results suggested that PERK activation may take place independent of the active site which can be blocked by a kinase inhibitor. The developed assay revealed to be sufficiently stable and robust to assess an activating EC 50-value. Instead of using PERK’s natural substrate proteins, eIF2α and NRF2, we applied SMAD3 as phosphorylation-accepting protein and successfully detected cell-free PERK activation and inhibition by selected modulators (e.g., calcineurin-B, GSK2606414). By applying the catalytic domain of recombinant human PERK, we initially determined ideal conditions of the kinase assay reaction, including parameters such as optimal kinase concentration, temperature, and reaction time. Our study aimed at the development of a cell-free screening assay enabling the detection of novel direct PERK activators. So far, a shortage of available direct PERK activators has been limiting the progresses in this field. The activation of the unfolded protein response, particularly via the PERK pathway, has been suggested as a promising therapeutic approach in tauopathies, a group of neurodegenerative disorders characterized by the abnormal phosphorylation and aggregation of tau protein.
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