Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress–induced hepatosteatosis

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1^S724A/S724A) and investigated the importance of phosphorylation at Ser⁷²⁴ within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser⁷²⁴ exacerbated ER stress–induced hepatic steatosis in tunicamycin-treated Ern1^S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer–binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser⁷²⁴ of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.     

靶向IRE1/XBP1信号通路的细胞水平高通量筛选模型建立

目的:建立基于细胞水平的inositol-requiring 1/X-box-binding protein 1 (IRE1/XBP1)信号通路高通量筛选模型,用于发现新型IRE1/XBP1信号通路抑制剂。方法:构建pCAX-F-XBP1△DBD-luciferase质粒,并与pcDNA3.1质粒共转人胚肾细胞HEK293,G418抗性筛选获得多个稳定表达荧光素酶的单克隆。结果:首先利用内质网应激诱导剂衣霉素(tunicamycin,TM)考察单克隆对内质网应激反应的敏感性,确定6#单克隆用于后续研究;其次对细胞接种量、溶剂DMSO终浓度和TM的作用浓度与孵育时间等条件进行优化,最终确定高通量筛选模型条件, Z'因子达到0.62;最后对包含多个激酶抑制剂在内的449个化合物进行筛选,发现27个潜在的IRE1/XBP1抑制剂,其中MG132、Sunitinib和Staurosporine的IC50分别为6.61(±1.51)μmol/L、6.25(±0.36)μmol/L和48(±8)nmol/L。结论:成功建立有效靶向IRE1/XBP1信号通路的高通量药物筛选模型,为基于IRE1/XBP1信号通路为靶点的药物发现奠定坚实基础。     

IRE1通路与肿瘤

内质网应激是细胞内广泛存在的一种应激反应。研究表明,内质网应激与肿瘤的发生发展密切相关。针对内质网应激及其相应信号通路进行肿瘤的预防或治疗受到了广泛关注。IRE1(inositol-requiring enzyme 1)通路是内质网应激诱发的最保守的信号通路。研究证实,IRE1及其主要的下游效应分子剪切型X 盒结合蛋白1与肿瘤进展密切相关。本文对IRE1通路与肿瘤发生发展、血管新生、肿瘤转移、肿瘤耐药性和恶性程度的相关性进行了阐述,同时分析了IRE1在不同肿瘤样本中的突变率、突变类型与病人存活状态的关系。作为肿瘤治疗的有效靶点,针对IRE1通路的调控能够有效延缓肿瘤的发生发展。     

Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor,IRE1β

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1α and IRE1β, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1β senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1β clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1β complex is robustly increased upon ER stress. Moreover, unlike IRE1α, the luminal domain of IRE1β showed anti-aggregation activity in vitro, and IRE1β was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1β, while IRE1α was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1α, the luminal event mediated by IRE1β involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.     

Cdc37/Hsp90 protein-mediated regulation of IRE1α protein activity in endoplasmic reticulum stress response and insulin synthesis in INS-1 cells

IRE1α is an endoplasmic reticulum (ER) localized signaling molecule critical for unfolded protein response. During ER stress, IRE1α activation is induced by oligomerization and autophosphorylation in its cytosolic domain, a process triggered by dissociation of an ER luminal chaperone, binding immunoglobulin-protein (BiP), from IRE1α. In addition, inhibition of a cytosolic chaperone protein Hsp90 also induces IRE1α oligomerization and activation in the absence of an ER stressor. Here, we report that the Hsp90 cochaperone Cdc37 directly interacts with IRE1α through a highly conserved cytosolic motif of IRE1α. Cdc37 knockdown or disruption of Cdc37 interaction with IRE1α significantly increased basal IRE1α activity. In INS-1 cells, Hsp90 inhibition and disruption of IRE1α-Cdc37 interaction both induced an ER stress response and impaired insulin synthesis and secretion. These data suggest that Cdc37-mediated direct interaction between Hsp90/Cdc37 and an IRE1α cytosolic motif is important to maintain basal IRE1α activity and contributes to normal protein homeostasis and unfolded protein response under physiological stimulation.     

Inositol-requiring protein 1 – X-box-binding protein 1 pathway promotes epithelial–mesenchymal transition via mediating snail expression in pulmonary fibrosis

Epithelial–mesenchymal transition (EMT) is a complex biological program during which cells loss epithelial phenotype and acquire mesenchymal features. EMT is thought to be involved in the pathogenesis of various fibrotic diseases including pulmonary fibrosis (PF). Recent studies suggest that endoplasmic reticulum (ER) stress is associated with EMT in the progression of PF. However, the exact mechanism is unclear. Here, we developed a PF model with bleomycin (BLM) administration in rats and conducted several simulation experiments in alveolar epithelial cell (AECs) RLE-6TN to unravel the role of inositol-requiring protein 1 (IRE1) – X-box-binding protein 1 (XBP1) signal pathway in ER stress-induced EMT in PF. First, we observed that ER stress was occurred in type II AECs accompanied by EMT in BLM-induced PF. Then we explored the role of IRE1-XBP1-snail pathway in transforming growth factor (TGF)-β1/tunicamycin (TM)-induced EMT. When TGF-β1/TM was treated on AECs, IRE1 and XBP1 were overexpressed, meanwhile, snail expression was upregulated accompanied with EMT. However, when IRE1 or XBP1 was knockdown, TGF-β1/TM-induced EMT were blocked while the expression of snail was inhibited. Then we silenced snail and found that TGF-β1/TM-induced EMT were also suppressed, but it had no effect on the up-regulated expression of IRE1 and XBP1. Thus, we concluded that IRE1-XBP1 pathway promotes EMT via mediating snail expression in PF.