肿瘤细胞中TMEM216 通过调控EIF2AK3影响内质网应激的分子机制

Endoplasmic reticulum stress plays critical role in apoptosis induced by many anti-cancer chemotherapeutic agents. As one of the receptors of HSPA5(BIP), EIF2AK3 is an important sensor protein in the ER membrane which triggers ER stress pathway. However, the mechanism underlying the regulation of EIF2AK3 remains elusive. Our preliminary data show that TMEM216 has physical interaction with EIF2AK3. In addition, E3 ligase SYVN1 binds to EIF2AK3 and elevates its ubiquitination and degradation. Therefore, we hypothesize that anti-cancer chemotherapeutic agents up-regulate TMEM216 transcriptionally. Moreover, TMEM216 binds to EIF2AK3, which enhances HSPA5 release from EIF2AK3, resulting in EIF2AK3 activation. TMEM216 also inhibits the recruitment of E3 ligase (e.g.SYVN1) to EIF2AK3, leading to reduction of its ubiquitination and degradation. Accordingly, TMEM216 promotes the apoptosis mediated by ER stress. Our goal is to characterize the roles of TMEM216 in the regulation of EIF2AK3 stability and activity in ER stress in cancer cells. Eventually, this study will help us understand the apoptotic signaling in cancer cells and potentially facilitate anti-cancer drug discovery and development.

多种化疗药物诱导内质网应激介导的肿瘤细胞凋亡。EIF2AK3（PERK）作为HSPA5（BIP）的受体之一，是内质网膜上重要的感受器蛋白，激活内质网应激通路，但其在内质网膜上的调控机制却并不清楚。我们在前期实验中发现跨膜蛋白TMEM216与EIF2AK3存在物理上的相互作用；抑制TMEM216表达增强EIF2AK3泛素化并下调其水平；E3连接酶SYVN1与EIF2AK3结合并影响其泛素化。据此提出假说：TMEM216与内质网膜上的感受器蛋白EIF2AK3存在相互作用，抑制HSPA5与EIF2AK3的结合，提高其活性；TMEM216也可抑制E3连接酶（如SYVN1）与EIF2AK3结合，降低EIF2AK3的泛素化和降解，提高其稳定性，最终促进内质网应激介导的肿瘤细胞凋亡。本项目拟确定TMEM216调控EIF2AK3活性及稳定性的分子机制，为开发新型抗肿瘤药物提供靶标和理论依据。

化疗药物通过内质网应激促进肿瘤细胞凋亡的研究已有很多报道，因此研究内质网应激对于了解肿瘤细胞凋亡调控机制。EIF2AK3作为重要的UPR感受器蛋白，关于其蛋白稳定性及降解机制的研究尚未见报道。TMEM216对维持细胞正常生理状态起着非常重要的作用。本课题以TMEM216为突破口，研究EIF2AK3的调控机制，阐明TMEM216调控内质网应激的分子机制，为寻找新的肿瘤治疗靶标提供理论依据。.不同NSCLC细胞中TMEM216和EIF2AK3的蛋白水平具有相关性；敲低TMEM216后，EIF2AK3及其下游相关蛋白下调，而对ERN1的蛋白水平没有影响；过表达TMEM216会上调 EIF2AK3及下游相关蛋白的水平。敲低TMEM216表达，WB和FACS显示细胞凋亡水平下调；过表达TMEM216后凋亡水平上调，表明TMEM216促进NSCLC细胞凋亡。敲低EIF2AK3后过表达TMEM216，发现敲低EIF2AK3抑制了TMEM216促凋亡效应，表明TMEM216通过上调EIF2AK3促进细胞凋亡。.TMEM216与EIF2AK3存在相互作用，调控EIF2AK3经泛素-蛋白酶体途径的降解。筛选出EIF2AK3一个可能的E3泛素连接酶SYVN1。数据表明TMEM216抑制EIF2AK3与SYVN1结合，并下调EIF2AK3的多聚泛素化水平。内质网应激可能会通过上调DDIT3调控TMEM216。E3泛素连接酶TRIM13也可以与EIF2AK3结合，上调EIF2AK3的泛素化水平促进其降解。.构建了Tmem216敲除小鼠，发现其具有纤毛病表型特征。Tmem216 敲除影响纤毛生成和初级纤毛的稳定性。.综上所述，NSCLC细胞中TMEM216上调EIF2AK3蛋白水平，促进细胞凋亡；TMEM216调控EIF2AK3经泛素-蛋白酶体途径的降解，SYVN1和TRIM13可能是EIF2AK3的两个E3连接酶；TMEM216抑制EIF2AK3与SYVN1或TRIM13相结合进而下调其多聚泛素化水平；内质网应激可能会通过上调DDIT3调控TMEM216；构建了TMEM216敲除小鼠，TMEM216 通过抑制 GLI2/GLI3 与 SUFU 的结合促进Hedgehog通路和纤毛形成。.本项目资助下已发表8篇SCI论文，另有2篇在审稿和投稿中。本项目培养博士生3名、硕士研究生4名。