BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy optio...BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.展开更多
Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the indu...Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.展开更多
目的鉴定1型登革病毒感染病人血清中1型登革病毒非结构蛋白1(nonstructural Protein 1 of Dengue virus type 1,DENV-1 NS1)B细胞线性表位。方法以重组DENV-1 NS1为抗原,免疫印迹法筛选NS1阳性病人血清。一组重叠DENV-1 NS1的重叠多肽...目的鉴定1型登革病毒感染病人血清中1型登革病毒非结构蛋白1(nonstructural Protein 1 of Dengue virus type 1,DENV-1 NS1)B细胞线性表位。方法以重组DENV-1 NS1为抗原,免疫印迹法筛选NS1阳性病人血清。一组重叠DENV-1 NS1的重叠多肽同这些NS1阳性血清反应鉴定NS1蛋白线性B细胞表位。采用梯度稀释的1-4型重组DENVNS1竞争抑制阳性多肽同病人血清反应鉴定其反应特异性和交叉反应性。结果12份DENV-1核酸阳性病人中筛选得到5份抗DENV-1 NS1阳性病人血清。重叠多肽法、竞争抑制法和生物信息学方法筛选出3条阳性NS1多肽(氨基酸残基序列第1-15,131-145,271-285),其中aa1-15在已报道的DENV-1分离株高度保守,提示这个区域可能存在DENV-1血清型特异性表位。aa131-145和aa271-285对应的蛋白序列比对分析表明以上区域中的133-FI/LIDGP-138和271-GKLEL/M/IDF-277在已报道的4种血清型DENV分离株中高度保守,提示这2个区域存在4种血清型DENV共同表位。结论发现3个NS1上B细胞线性表位,其中aa131-145和aa271-285为首次报道,结果有助于疫苗和诊断试剂的研发。展开更多
Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the cr...Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain （RBD） bound to a double-stranded RNA （dsRNA） at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel a-helices, dsRNA is anchored by a pair of invariable arginines （Arg38） from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.展开更多
基金the National Key Research and Development Program of China,No.2017YFC0908104National Science and Technology Projects,No.2017ZX10203201,No.2017ZX10201201,and No.2017ZX10202202.
文摘BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.
基金Supported by the National Natural Science Foundation of China(No.30671852)the Open Research Fund Program of the State Key Laboratory of Virology of China(Nos.2010009, 2007007) the Research Fund of the Key Laboratory of Department of Education of Liaoning Province, China(No.2009S043)
文摘Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.
文摘目的鉴定1型登革病毒感染病人血清中1型登革病毒非结构蛋白1(nonstructural Protein 1 of Dengue virus type 1,DENV-1 NS1)B细胞线性表位。方法以重组DENV-1 NS1为抗原,免疫印迹法筛选NS1阳性病人血清。一组重叠DENV-1 NS1的重叠多肽同这些NS1阳性血清反应鉴定NS1蛋白线性B细胞表位。采用梯度稀释的1-4型重组DENVNS1竞争抑制阳性多肽同病人血清反应鉴定其反应特异性和交叉反应性。结果12份DENV-1核酸阳性病人中筛选得到5份抗DENV-1 NS1阳性病人血清。重叠多肽法、竞争抑制法和生物信息学方法筛选出3条阳性NS1多肽(氨基酸残基序列第1-15,131-145,271-285),其中aa1-15在已报道的DENV-1分离株高度保守,提示这个区域可能存在DENV-1血清型特异性表位。aa131-145和aa271-285对应的蛋白序列比对分析表明以上区域中的133-FI/LIDGP-138和271-GKLEL/M/IDF-277在已报道的4种血清型DENV分离株中高度保守,提示这2个区域存在4种血清型DENV共同表位。结论发现3个NS1上B细胞线性表位,其中aa131-145和aa271-285为首次报道,结果有助于疫苗和诊断试剂的研发。
文摘Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain （RBD） bound to a double-stranded RNA （dsRNA） at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel a-helices, dsRNA is anchored by a pair of invariable arginines （Arg38） from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.
