棒状体相关蛋白1(rhoptry-associated protein 1,RAP-1)是由棒状体分泌的与侵袭宿主细胞相关的蛋白。本研究以顶复门原虫RAP-1基因为靶标,用最大似然法构建了巴贝斯属、疟原虫属、泰勒虫属的RAP-1基因进化树;应用软件预测分析RAP-1蛋白...棒状体相关蛋白1(rhoptry-associated protein 1,RAP-1)是由棒状体分泌的与侵袭宿主细胞相关的蛋白。本研究以顶复门原虫RAP-1基因为靶标,用最大似然法构建了巴贝斯属、疟原虫属、泰勒虫属的RAP-1基因进化树;应用软件预测分析RAP-1蛋白的理化性质、亲疏水性、跨膜区以及二、三级结构,并表达纯化了马泰勒虫(Theileria equi)RAP-1重组蛋白。结果显示:巴贝斯属不同虫株相聚类,测定序列与马泰勒虫聚为一支,又与恶性疟原虫相聚类,表明亲缘关系较近。马泰勒虫RAP-1蛋白理论等电点为9.85,半衰期为30 h,总平均亲水性(GRAVY)为-0.368,无跨膜区;RAP-1蛋白二级结构中,α-螺旋、β-折叠、β-转角和无规卷曲占比分别为38.5%、15.5%、27.8%、18.1%;构建的三级结构立体展现了RAP-1蛋白形态。成功构建了原核表达载体pET-32a-RAP-1;该重组蛋白主要以包涵体形式存在,蛋白大小为65 kDa。RAP-1蛋白可通过原核表达系统高效表达,纯化后的产物能被马泰勒虫标准阳性血清识别,具有良好的反应原性。本研究为深入了解马泰勒虫入侵宿主的机制机理以及RAP-1蛋白的功能研究奠定了基础。展开更多
Rapl is expressed in human umbilical vein endothelial cells （HUVECs）. Rapl-GTPase activating protein （RaplGAP）, with its specific target, Rapl, has been shown to be important in the regulation of many physiologica...Rapl is expressed in human umbilical vein endothelial cells （HUVECs）. Rapl-GTPase activating protein （RaplGAP）, with its specific target, Rapl, has been shown to be important in the regulation of many physiological and certain pathological processes. In this study, we investigated the effect of RaplGAP expression on endothelial cell function, or, more specifically, proliferation and migration of endothelial cells. HUVECs were transfected with pcDNA3.1 （empty vector）, pcDNA3.1 containing Flag-tagged-RaplGAP or Myc-tagged-RaplN17. The proliferation, migration and tube formation were examined and compared among the 3 groups. Expression of Rapl, RaplGAP, extracellular signal-regulated kinase （ERK）, phospho-ERK, Akt, phosphor-Akt was detected by Western blotting. The results showed that the proliferation, migration and tube formation were significantly reduced in RaplGAP- and RaplN17-transfected HUVECs as compared with empty vector-transfected control. These changes were coincident with increased expression of Rap 1GAP and decreased expression of activated Rap l, phospho-ERK and -Akt. After treatment of Rap l GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor （RaplGEF） 8CPT-2＇OMe-cAMP, it was found that Rapl activity was decreased as compared with empty vector-transfected control. Pretreatment of HU- VECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2＇OMe-cAMP-induced phosphorylation of ERK and Akt, but also significantly reduced cell proliferation and migration. Finally, we examined the effect of vascular endothelial growth factor （VEGF） on HUVECs overexpressing RaplGAP. VEGF-stimulated Rapl activity, phosphorylation of ERK and Akt, cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing RaplGAP as compared to empty vector-transfected Control. Taken together, our findings demonstrate that RaplGAP/Rapl and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.展开更多
Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)spe...Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.30971207)Natural Science Foundation of Hubei Province,China(No.2009CBD-386)
文摘Rapl is expressed in human umbilical vein endothelial cells （HUVECs）. Rapl-GTPase activating protein （RaplGAP）, with its specific target, Rapl, has been shown to be important in the regulation of many physiological and certain pathological processes. In this study, we investigated the effect of RaplGAP expression on endothelial cell function, or, more specifically, proliferation and migration of endothelial cells. HUVECs were transfected with pcDNA3.1 （empty vector）, pcDNA3.1 containing Flag-tagged-RaplGAP or Myc-tagged-RaplN17. The proliferation, migration and tube formation were examined and compared among the 3 groups. Expression of Rapl, RaplGAP, extracellular signal-regulated kinase （ERK）, phospho-ERK, Akt, phosphor-Akt was detected by Western blotting. The results showed that the proliferation, migration and tube formation were significantly reduced in RaplGAP- and RaplN17-transfected HUVECs as compared with empty vector-transfected control. These changes were coincident with increased expression of Rap 1GAP and decreased expression of activated Rap l, phospho-ERK and -Akt. After treatment of Rap l GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor （RaplGEF） 8CPT-2＇OMe-cAMP, it was found that Rapl activity was decreased as compared with empty vector-transfected control. Pretreatment of HU- VECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2＇OMe-cAMP-induced phosphorylation of ERK and Akt, but also significantly reduced cell proliferation and migration. Finally, we examined the effect of vascular endothelial growth factor （VEGF） on HUVECs overexpressing RaplGAP. VEGF-stimulated Rapl activity, phosphorylation of ERK and Akt, cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing RaplGAP as compared to empty vector-transfected Control. Taken together, our findings demonstrate that RaplGAP/Rapl and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.
基金supported by National Natural Science Foundation of China(Grant Number 81472261)Natural Science Foundation of Guangdong Province(Grant Numbers 2014A030310033 and 2015A030313032)+1 种基金Science and Technology Planning Project of Guangdong Province(Grant Number 2013B021800122)Science and Technology Planning Projects of Guangzhou City(Grant Number 201604020044).
文摘Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.
