Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. Comprehensive transcriptome analysis was employed to validate the capacity of three prioritized compounds to remodel the ER proteostasis environment, and to assess the prefential activation of ATF6 transcriptional targets relative to targets of the IRE1/XBP1s and PERK arms of the UPR. HEK293T-Rex and HEK293-DAX cells were treated for 6 hr with vehicle (DMSO), 1 µM Tg, 10 mM TMP (in HEK293DAX), or 10 µM 132, 147 or 263 in biological triplicate at 37 ̊C

内质网（endoplasmic reticulum, ER）蛋白质稳态（proteostasis）失衡与病因各异的退行性疾病密切相关，此类疾病多伴随过量细胞外蛋白质错误折叠与聚集。通过对未折叠蛋白反应（Unfolded Protein Response, UPR）相关转录激活因子6（ATF6）进行遗传激活以重编程内质网蛋白质稳态环境，可有效抑制淀粉样蛋白的分泌及细胞外聚集。本研究采用了一套整合互补分支特异性UPR报告基因系统与中通量转录谱分析的筛选策略，旨在筛选出能够模拟ATF6介导的内质网蛋白质稳态重编程过程的无毒小分子化合物。随后通过全转录组分析，验证了3种优先候选化合物重塑内质网蛋白质稳态的能力，并对比UPR的IRE1/XBP1s与PERK分支靶点，评估了ATF6转录靶点的优先激活效应。实验中将HEK293T-Rex与HEK293-DAX细胞置于37℃环境，分别以溶剂对照（二甲基亚砜, DMSO）、1 μM毒胡萝卜素（Tg）、10 mM TMP（仅用于HEK293DAX细胞）以及10 μM的132、147或263处理6小时，每组设置生物学重复三次。