Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to PKM2-dependent conventional caspase-3/GSDME cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-PD-1. In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.

中文翻译：

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通过靶向 RTN4 进行内质网膜重塑可诱导细胞焦亡，以促进抗肿瘤免疫。


细胞焦亡是一种已确定的程序性细胞死亡，与内质网 (ER) 动力学高度相关。然而，调节内质网膜曲率动态变化（引发细胞焦亡）的关键蛋白目前尚不清楚。在本研究中，合成了有效焦亡诱导剂α-山竹素（α-MG）的生物素标记化学探针。通过蛋白质微阵列分析，内质网膜曲率的关键调节因子 reticulon-4 (RTN4/Nogo) 被确定为 α-MG 的靶标。我们观察到，α-MG 通过招募 E3 连接酶 UBR5 化学诱导 RTN4 的蛋白酶体降解，显着增强癌细胞的焦亡表型。有趣的是，RTN4表达的下调显着促进了内质网膜曲率的动态重塑，从小管到片层的转变，从而导致内质网与细胞质膜的快速融合。特别是，观察到的几个关键的内质网标记易位到焦亡细胞的“气泡”结构，支持了内质网与质膜的融合过程。此外，α-MG 诱导的 RTN4 敲低会导致 PKM2 依赖性的常规 caspase-3/GSDME 裂解，从而促进焦亡进展。在体内，我们观察到化学或基因敲除 RTN4 显着抑制癌细胞生长，从而进一步表现出抗 PD-1 的抗肿瘤免疫反应。在转化研究中，RTN4高表达与肿瘤转移和患者死亡密切相关。总而言之，RTN4 通过调节 ER 膜曲率重塑在诱导细胞焦亡中发挥着重要作用，因此代表了抗癌免疫治疗的潜在药物靶标。