目的探讨α吡喃酮类化合物Rasfonin对鸟苷酸交换因子SOS1表达的调控作用及其机制。方法(1)将MCF-7、Calu-1和UM-UC-3细胞分别分为溶剂对照组和Rasfonin组(1、5、10和15μmol·L^(-1)),处理24 h后CCK-8法检测MCF-7、Calu-1和UM-UC-3...目的探讨α吡喃酮类化合物Rasfonin对鸟苷酸交换因子SOS1表达的调控作用及其机制。方法(1)将MCF-7、Calu-1和UM-UC-3细胞分别分为溶剂对照组和Rasfonin组(1、5、10和15μmol·L^(-1)),处理24 h后CCK-8法检测MCF-7、Calu-1和UM-UC-3细胞存活率。将MCF-7、Calu-1和UM-UC-3细胞分别分为细胞对照组(培养基)、表皮生长因子组(EGF 50μg·L^(-1)处理5 min),EGF+Rasfonin组(Rasfonin分别以5和10μmol·L^(-1)预处理不同时间后,再EGF 50μg·L^(-1)处理5 min),实时荧光定量PCR和Western印迹法检测MCF-7、Calu-1和UM-UC-3细胞中SOS1 m RNA和蛋白表达水平。(2)将阴性对照(NC)质粒、KRASWT质粒和KRASG12C质粒分别与SOS1质粒共转染至293T细胞,转染后分为溶剂对照组和Rasfonin组(1、5和10μmol·L^(-1)),处理12 h后采用双荧光素酶报告基因实验检测SOS1启动子活性。将质粒KRAS^(WT)、KRAS^(G12C)、NC+SOS1、KRAS^(WT)+SOS1和KRAS^(G12C)+SOS1分别转染至293T细胞,分为EGF组和EGF+Rasfonin组(Rasfonin 10μmol·L^(-1)预处理12 h后,再EGF处理5 min),Western印迹法检测293T细胞中SOS1蛋白表达水平。结果(1)与溶剂对照组相比,Rasfonin 5、10和15μmol·L^(-1)显著抑制Calu-1和UM-UC-3细胞增殖,IC_(50)分别为8.22和4.94μmol·L^(-1),MCF-7细胞的IC50为45.15μmol·L^(-1)。Rasfonin处理3 h MCF-7细胞SOS1 m RNA水平升高;Rasfonin处理1 h Calu-1细胞SOS1 m RNA水平升高,3和6 h SOS1 m RNA水平降低;Rasfonin处理3 h UM-UC-3细胞SOS1 m RNA表达,6 h降低。与EGF组相比,EGF+Rasfonin组MCF-7细胞的SOS1蛋白表达水平无显著变化,Calu-1与UM-UC-3细胞的SOS1蛋白表达水平显著降低。(2)与溶剂对照组相比,Rasfonin对SOS1单表达组SOS1启动子活性无显著影响,但SOS1与KRAS^(WT)或KRAS^(G12C)蛋白共表达时,SOS1启动子活性被Rasfonin显著抑制。与EGF组相比,Rasfonin对SOS1单表达组及SOS1+KRAS^(WT)共表达组的SOS1蛋白表达水平无显著变化,SOS1+KRAS^(G12C)共表达组的SOS1蛋白表达水平显著降低。结论Rasfonin通过KRASG12C依赖性途径抑制SOS1表达,这可能是其抗肿瘤作用的机制之一。展开更多
Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos i...Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos is regulated by its interaction with Ras·GTP at the allosteric site of Sos.Although much research has been conducted,the structural details of the dynamic interactions between Ras·GTP and Sos at the allosteric site in solution are not completely understood.In this study,paramagnetic relaxation enhancement(PRE)was used to examine the binding of HRas·GTP to the allosteric site of Soscat.A site-specific paramagnetic probe was attached to Soscat,revealing crucial interface information,which was then used to build structural models of the HRas-Soscat complex via HADDOCK.Our results indicate that the dominant conformation of HRas·GTP bound to Soscat at the allosteric site is highly consistent with the established crystal structure while also identifying a transient conformation with a novel interface between HRas and Sos unseen in previous crystal structures.展开更多
针对一类由众多组件系统集结而成的系统之系统(system of systems,SoS),以美国国防部体系结构框架(DoDAF)为标准,提出了一种基于面向对象思想的SoS体系结构DoDAF作战视图产品五阶段迭代设计方法。利用UML静态和动态建模机制的特点,采用...针对一类由众多组件系统集结而成的系统之系统(system of systems,SoS),以美国国防部体系结构框架(DoDAF)为标准,提出了一种基于面向对象思想的SoS体系结构DoDAF作战视图产品五阶段迭代设计方法。利用UML静态和动态建模机制的特点,采用自顶向下、自底向上相结合的方式实现SoS体系结构作战视图产品的面向对象描述。以一个战术导弹防御(tactical missile defense,TMD)系统为例,详细说明SoS体系结构DoDAF作战视图产品的面向对象设计过程,并总结了该方法的两大优越特性:横向通用性与纵向可复用性,以及由此给SoS体系结构带来的"柔性"优势。这是传统设计方法所不能实现的。展开更多
文摘目的探讨α吡喃酮类化合物Rasfonin对鸟苷酸交换因子SOS1表达的调控作用及其机制。方法(1)将MCF-7、Calu-1和UM-UC-3细胞分别分为溶剂对照组和Rasfonin组(1、5、10和15μmol·L^(-1)),处理24 h后CCK-8法检测MCF-7、Calu-1和UM-UC-3细胞存活率。将MCF-7、Calu-1和UM-UC-3细胞分别分为细胞对照组(培养基)、表皮生长因子组(EGF 50μg·L^(-1)处理5 min),EGF+Rasfonin组(Rasfonin分别以5和10μmol·L^(-1)预处理不同时间后,再EGF 50μg·L^(-1)处理5 min),实时荧光定量PCR和Western印迹法检测MCF-7、Calu-1和UM-UC-3细胞中SOS1 m RNA和蛋白表达水平。(2)将阴性对照(NC)质粒、KRASWT质粒和KRASG12C质粒分别与SOS1质粒共转染至293T细胞,转染后分为溶剂对照组和Rasfonin组(1、5和10μmol·L^(-1)),处理12 h后采用双荧光素酶报告基因实验检测SOS1启动子活性。将质粒KRAS^(WT)、KRAS^(G12C)、NC+SOS1、KRAS^(WT)+SOS1和KRAS^(G12C)+SOS1分别转染至293T细胞,分为EGF组和EGF+Rasfonin组(Rasfonin 10μmol·L^(-1)预处理12 h后,再EGF处理5 min),Western印迹法检测293T细胞中SOS1蛋白表达水平。结果(1)与溶剂对照组相比,Rasfonin 5、10和15μmol·L^(-1)显著抑制Calu-1和UM-UC-3细胞增殖,IC_(50)分别为8.22和4.94μmol·L^(-1),MCF-7细胞的IC50为45.15μmol·L^(-1)。Rasfonin处理3 h MCF-7细胞SOS1 m RNA水平升高;Rasfonin处理1 h Calu-1细胞SOS1 m RNA水平升高,3和6 h SOS1 m RNA水平降低;Rasfonin处理3 h UM-UC-3细胞SOS1 m RNA表达,6 h降低。与EGF组相比,EGF+Rasfonin组MCF-7细胞的SOS1蛋白表达水平无显著变化,Calu-1与UM-UC-3细胞的SOS1蛋白表达水平显著降低。(2)与溶剂对照组相比,Rasfonin对SOS1单表达组SOS1启动子活性无显著影响,但SOS1与KRAS^(WT)或KRAS^(G12C)蛋白共表达时,SOS1启动子活性被Rasfonin显著抑制。与EGF组相比,Rasfonin对SOS1单表达组及SOS1+KRAS^(WT)共表达组的SOS1蛋白表达水平无显著变化,SOS1+KRAS^(G12C)共表达组的SOS1蛋白表达水平显著降低。结论Rasfonin通过KRASG12C依赖性途径抑制SOS1表达,这可能是其抗肿瘤作用的机制之一。
基金supported by the Fundamental Research Funds for the Central Universities(WK9100000046).
文摘Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos is regulated by its interaction with Ras·GTP at the allosteric site of Sos.Although much research has been conducted,the structural details of the dynamic interactions between Ras·GTP and Sos at the allosteric site in solution are not completely understood.In this study,paramagnetic relaxation enhancement(PRE)was used to examine the binding of HRas·GTP to the allosteric site of Soscat.A site-specific paramagnetic probe was attached to Soscat,revealing crucial interface information,which was then used to build structural models of the HRas-Soscat complex via HADDOCK.Our results indicate that the dominant conformation of HRas·GTP bound to Soscat at the allosteric site is highly consistent with the established crystal structure while also identifying a transient conformation with a novel interface between HRas and Sos unseen in previous crystal structures.
文摘针对一类由众多组件系统集结而成的系统之系统(system of systems,SoS),以美国国防部体系结构框架(DoDAF)为标准,提出了一种基于面向对象思想的SoS体系结构DoDAF作战视图产品五阶段迭代设计方法。利用UML静态和动态建模机制的特点,采用自顶向下、自底向上相结合的方式实现SoS体系结构作战视图产品的面向对象描述。以一个战术导弹防御(tactical missile defense,TMD)系统为例,详细说明SoS体系结构DoDAF作战视图产品的面向对象设计过程,并总结了该方法的两大优越特性:横向通用性与纵向可复用性,以及由此给SoS体系结构带来的"柔性"优势。这是传统设计方法所不能实现的。
