蛋白质-蛋白质相互作用在细胞的生化功能中扮演极为重要的角色,深入解析蛋白质相互作用关系是理解细胞生命活动的关键。本研究以高尔基体蛋白73(golgi protein 73,GP73)为研究对象,利用经典的免疫共沉淀联合质谱技术系统挖掘了GP73的相...蛋白质-蛋白质相互作用在细胞的生化功能中扮演极为重要的角色,深入解析蛋白质相互作用关系是理解细胞生命活动的关键。本研究以高尔基体蛋白73(golgi protein 73,GP73)为研究对象,利用经典的免疫共沉淀联合质谱技术系统挖掘了GP73的相互作用蛋白质,力求进一步解析GP73的分子功能。选取肝癌细胞系HepG2,利用慢病毒感染技术构建过表达GP73-3Flag的稳定细胞系,免疫共沉淀联合质谱检测鉴定出78个高置信的GP73相互作用蛋白质,生物信息学分析提示,GP73与近40个细胞核蛋白质存在相互作用,并参与RNA运输、剪接和翻译等生物学过程,进一步的免疫荧光和细胞核蛋白质分离实验证实,GP73在多种肿瘤细胞中的细胞核定位,在78个相互作用蛋白质的基础上进一步筛选出与mRNA剪接相关的蛋白质相互作用网络,并通过免疫共沉淀验证了GP73与HNRNPH3、SMN1、RBM14、NCBP1等7种蛋白质存在相互作用。Minigene剪接实验提示,过表达GP73抑制细胞对pre-mRNA的剪接效率。本研究拓展了对GP73蛋白功能的认识和理解,有助于解释其在细胞生物学中的重要角色及其与疾病的潜在关联。展开更多
Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to ...Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role.Methods Initially,we assessed PES1 expression patterns in two distinct senescence models:replicative senescent mouse embryonic fibroblasts(MEFs)and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells.Subsequently,PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types.Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays,respectively.The expression of senescence-associated proteins(p53,p21,and Rb)and SASP factors(IL-6,IL-1β,and IL-8)were analyzed by Western blot or qPCR.Furthermore,Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology.Results PES1 expression was significantly downregulated in senescent MEFs and HepG2 cells.PES1 knockdown resulted in decreased EdU-positive cells and increased SA-β-gal-positive cells,indicating proliferation inhibition and senescence induction.Mechanistically,PES1 suppression activated the p53-p21 pathway without affecting Rb expression,while upregulating IL-6,IL-1β,and IL-8 production.Notably,PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress,as evidenced by aberrant nucleolar morphology.Conclusion Our findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent(but Rb-independent)cellular senescence,highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.展开更多
文摘蛋白质-蛋白质相互作用在细胞的生化功能中扮演极为重要的角色,深入解析蛋白质相互作用关系是理解细胞生命活动的关键。本研究以高尔基体蛋白73(golgi protein 73,GP73)为研究对象,利用经典的免疫共沉淀联合质谱技术系统挖掘了GP73的相互作用蛋白质,力求进一步解析GP73的分子功能。选取肝癌细胞系HepG2,利用慢病毒感染技术构建过表达GP73-3Flag的稳定细胞系,免疫共沉淀联合质谱检测鉴定出78个高置信的GP73相互作用蛋白质,生物信息学分析提示,GP73与近40个细胞核蛋白质存在相互作用,并参与RNA运输、剪接和翻译等生物学过程,进一步的免疫荧光和细胞核蛋白质分离实验证实,GP73在多种肿瘤细胞中的细胞核定位,在78个相互作用蛋白质的基础上进一步筛选出与mRNA剪接相关的蛋白质相互作用网络,并通过免疫共沉淀验证了GP73与HNRNPH3、SMN1、RBM14、NCBP1等7种蛋白质存在相互作用。Minigene剪接实验提示,过表达GP73抑制细胞对pre-mRNA的剪接效率。本研究拓展了对GP73蛋白功能的认识和理解,有助于解释其在细胞生物学中的重要角色及其与疾病的潜在关联。
文摘Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role.Methods Initially,we assessed PES1 expression patterns in two distinct senescence models:replicative senescent mouse embryonic fibroblasts(MEFs)and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells.Subsequently,PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types.Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays,respectively.The expression of senescence-associated proteins(p53,p21,and Rb)and SASP factors(IL-6,IL-1β,and IL-8)were analyzed by Western blot or qPCR.Furthermore,Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology.Results PES1 expression was significantly downregulated in senescent MEFs and HepG2 cells.PES1 knockdown resulted in decreased EdU-positive cells and increased SA-β-gal-positive cells,indicating proliferation inhibition and senescence induction.Mechanistically,PES1 suppression activated the p53-p21 pathway without affecting Rb expression,while upregulating IL-6,IL-1β,and IL-8 production.Notably,PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress,as evidenced by aberrant nucleolar morphology.Conclusion Our findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent(but Rb-independent)cellular senescence,highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.
