The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacA^u into its active form, hacAⁱ, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4μ8C prevented A. fumigatus from increasing the levels of hacAⁱ mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4μ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4μ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4μ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAⁱ mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacA^u mRNA in ER stress response.

中文翻译：

---

人类 IRE1 抑制剂阻断病原真菌烟曲霉中未折叠的蛋白质反应并暗示该途径中的非经典功能

未折叠蛋白反应 (UPR) 是一种信号网络，可维持内质网 (ER) 的稳态。在人类致病真菌烟曲霉中，UPR 是通过激活内质网跨膜应力传感器 IreA 中的核糖核酸内切酶 (RNase) 域而启动的，该域将下游 mRNA hacA ^u剪接成其活性形式hacA ⁱ，编码主转录调节因子的途径。针对 IRE1（IreA 的人类直系同源物）的小分子抑制剂已被开发用于抗癌治疗，但它们对真菌 UPR 的影响尚待探索。在这里，我们证明IRE1 RNA酶抑制剂4μ8C防止烟曲霉从提高水平hacA ⁱ mRNA，从而阻断下游 UPR 靶基因表达的诱导。4μ8C 处理对基本培养基中的生长影响很小，但严重损害了需要高水平水解酶分泌的胶原底物的生长，反映了其他真菌 UPR 突变体的表型。4μ8C 还增加了对香芹酚（一种破坏真菌内质网完整性的天然化合物）和潮霉素 B 的敏感性，后者与糖基化相关基因的表达减少有关。有趣的是，用 4μ8C 处理无法诱导归因于 Δ hacA突变体中经典 UPR 丢失的所有表型，但显示出与 RNase 缺陷型IreA突变体的显着相似性，该突变体也无法产生hacA^我是mRNA。这些结果证明了经典 UPR 途径的药理学抑制在烟曲霉中是可行的，并支持hacA ^u mRNA 在 ER 应激反应中的非经典作用。