A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.
A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.
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DOI：
10.1128/msphere.00879-20
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发表时间：
2020-10-21
期刊：
影响因子：
4.8
通讯作者：
Askew DS
中科院分区：
文献类型：
--
作者：
Guirao-Abad JP;Weichert M;Albee A;Deck K;Askew DS
未折叠蛋白反应（UPR）是一种维持内质网（ER）稳态的信号通路，其功能与人类致病性烟曲霉的毒力机制重叠。典型途径以HacA为中心，它是主要的转录调节因子。该蛋白的翻译需要从hacA基因的细胞质mRNA中去除一个非常规的内含子，这是通过位于er跨膜应力传感器IreA中的RNase结构域实现的。在这里，我们展示了用一种小分子抑制剂靶向这种RNase活性，有效地阻断了UPR的激活，从而产生了反映RNase结构域遗传缺失后果的效果。然而，与hacA基因完全缺失相关的表型相比，这些表型的范围令人惊讶地狭窄。这些发现通过支持未剪接的hacA mRNA在内质网应激反应中存在非规范功能，扩展了对该物种UPR信号传导的理解。未折叠蛋白反应（UPR）是维持内质网（ER）稳态的信号网络。在人类致病性真菌烟曲霉中，UPR是通过内质网跨膜应激传感器IreA中的核糖核酸内切酶（RNase）区域的激活而启动的，该区域将下游mRNA hacAu剪切成其活性形式hacAi，编码该途径的主转录调节因子。针对IRE1 （IRE1的人类同源物）的小分子抑制剂已被开发用于抗癌治疗，但其对真菌UPR的作用尚未探索。在这里，我们证明了IRE1 RNase抑制剂4μ8C阻止烟曲霉增加hacAi mRNA水平，从而阻断下游UPR靶基因表达的诱导。4μ8C处理对在最小培养基中的生长影响最小，但严重损害了在需要高水平水解酶分泌的胶原底物上的生长，反映了其他真菌UPR突变体的表型。4μ8C还增加了对carvacrol（一种破坏真菌内质网完整性的天然化合物）和hygromycin B（与糖基化相关基因表达减少相关）的敏感性。有趣的是，4μ8C处理无法诱导ΔhacA突变体中由于典型UPR缺失而导致的所有表型，但与同样无法产生hacAi mRNA的rnase缺陷IreA突变体的表型显着相似。这些结果证实了烟曲霉典型UPR通路的药理抑制是可行的，并支持hacAu mRNA在内质网应激反应中的非典型作用。未折叠蛋白反应（UPR）是一种维持内质网（ER）稳态的信号通路，其功能与人类致病性烟曲霉的毒力机制重叠。典型途径以HacA为中心，它是主要的转录调节因子。该蛋白的翻译需要从hacA基因的细胞质mRNA中去除一个非常规的内含子，这是通过位于er跨膜应力传感器IreA中的RNase结构域实现的。在这里，我们展示了用一种小分子抑制剂靶向这种RNase活性，有效地阻断了UPR的激活，从而产生了反映RNase结构域遗传缺失后果的效果。然而，与hacA基因完全缺失相关的表型相比，这些表型的范围令人惊讶地狭窄。这些发现通过支持未剪接的hacA mRNA在内质网应激反应中存在非规范功能，扩展了对该物种UPR信号传导的理解。
The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus. The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response. 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 hacAu 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 hacAu mRNA in ER stress response. IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus. The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.