同型半胱氨酸诱导的内质网蛋白(HERP)是内质网(ER)驻留蛋白,对于ER相关降解(ERAD)系统适应细胞蛋白质稳态非常重要。HERP相互作用剂对于细胞活力和对ER应激的反应至关重要。为了探索HERP发挥生物学功能的确切机制,我们通过免疫共沉淀(Co-IP)和液相色谱-质谱仪(LC-MS)/MS结合无标记定量(LFQ)对HeLa细胞中的HERP蛋白进行了相互作用分析.在相互作用组结果中,123个蛋白质与HERP显著相互作用,这导致了许多生物过程,包括蛋白质导入细胞核,泛素依赖性ERAD通路,凋亡过程的负调节,和来自ER的蛋白质转运,以及多种途径,包括几种疾病,ER中的蛋白质加工,脂肪酸代谢,和类固醇生物合成。此外,我们从相互作用组数据中选择了几种猎物蛋白,并证实了HERP与古老的普遍存在蛋白1(AUP1)相互作用,Fas相关因子家族成员2(FAF2),包含47(TRIM47)的三方基序,酰基辅酶A合成酶长链家族成员3(ACSL3),隔离体1(SQSTM1),和聚(rC)结合蛋白2(PCBP2)通过Co-IP和共聚焦显微镜实验,分别。此外,在Thapsigargin刺激和肠道病毒71型感染诱导的内质网应激下,几种相互作用蛋白的表达和位置发生了明显改变。总之,我们的发现表明,重要的蛋白质与HERP相互作用以介导信号转导,从而为在生理和病理条件下与ERAD和代谢相关的HERP响应ER应激的机制提供了新的线索。
Homocysteine-inducible endoplasmic reticulum protein (HERP) is an endoplasmic reticulum (ER)-resident protein and important for the adaptation of cellular protein homeostasis by ER-associated degradation (ERAD) system. HERP interactors are critical for cellular viability and the reaction to ER stress. To explore the exact mechanisms by which HERP performed the biological functions, we conducted an interaction analysis of HERP protein in HeLa cells by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometer (LC-MS)/MS coupled with label-free quantification (LFQ). Among the interactome results, 123 proteins significantly interacted with HERP, which leads to numerous biological processes including protein import into nucleus, ubiquitin-dependent ERAD pathway, negative regulation of apoptotic process, and protein transport from ER, along with multiple pathways including several diseases, protein processing in ER, fatty acid metabolism, and steroid biosynthesis. Furthermore, we selected several prey proteins from the interactome data and confirmed that HERP interacted with ancient ubiquitous protein 1 (AUP1), Fas-associated factor family member 2 (FAF2), tripartite motif containing 47 (TRIM47), acyl-CoA synthetase long-chain family member 3 (ACSL3), sequestosome 1 (SQSTM1), and poly(rC) binding protein 2 (PCBP2) by Co-IP and confocal microscopy experiments, respectively. Moreover, the expression and location of several interacted proteins were obviously altered in response to ER stress induced by Thapsigargin stimulation and Enterovirus 71 infection. In conclusion, our findings revealed that the vital proteins interacted with HERP to mediate signaling transduction, thus providing novel clues for the mechanisms of HERP associated with ERAD and metabolism in response to ER stress under physiological and pathological conditions.