Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE co...Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.展开更多
AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE v...AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.展开更多
目的构建通用流感mRNA疫苗并全面评价其免疫保护效果。方法优化流感病毒株A/California/04/2009的血凝素(hemagglutinin,HA)、核蛋白(nucleoprotein,NP)和基质蛋白2胞外区(matrix protein 2 ectodomain,M2e)抗原序列,并将HA、NP和3个串...目的构建通用流感mRNA疫苗并全面评价其免疫保护效果。方法优化流感病毒株A/California/04/2009的血凝素(hemagglutinin,HA)、核蛋白(nucleoprotein,NP)和基质蛋白2胞外区(matrix protein 2 ectodomain,M2e)抗原序列,并将HA、NP和3个串联的M2e(3M2e)分别克隆至pcDNA3.1载体,通过线性化、体外转录、酶学法加帽、酶促加尾合成mRNA,命名为mRNA-HA、mRNA-NP和mRNA-3M2e。3种mRNA分别转染293T细胞后,通过免疫荧光实验鉴定蛋白的表达。利用脂质纳米颗粒分别包裹mRNA-HA、mRNA-NP和mRNA-3M2e并测量其粒径和电位。再将3种mRNA等体积混合制备成Comb-mRNA疫苗。将28只6周雌性Balb/c小鼠(体质量为18~22 g)按简单随机分组法分为2组:LNP组(n=14)和Comb-mRNA组(n=14)。通过血凝抑制(hemagglutination inhibition,HI)、微量中和(microneutralization,MN)实验评价流感mRNA疫苗诱导小鼠产生的血清抗体滴度;通过流式细胞术评价Comb-mRNA疫苗诱导的细胞免疫反应。采用5LD_(50)野生型H1N1流感病毒株感染小鼠评价Comb-mRNA疫苗的免疫保护效果。结果成功构建mRNA-HA、mRNA-NP和mRNA-3M2e,且3种mRNA均能在293T细胞表达。脂质纳米颗粒包裹mRNA平均粒径为(119.53±6.5)nm,平均电位为(-8.23±1.3)mV。与LNP组比较,Comb-mRNA组疫苗的HI几何平均滴度(geometric mean titer,GMT)达179.6,MN的GMT达201.6,同时诱导IFNγ+CD4+/CD8+T细胞比例升高。Comb-mRNA组在加强免疫后2周能够提供对5LD_(50)野生流感H1N1亚型病毒的保护。结论通用流感疫苗候选疫苗Comb-mRNA能够诱导小鼠免疫反应并保护小鼠免受病毒感染。展开更多
Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used t...Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.展开更多
Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （T...Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （TS00） and full-length ectodomain （T1300） of TACE were amplified by RT-PCR, and the expres.sion plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant TS00 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that TS00 and T1300 were highly expressed in the form of inclusion body. After Ni^2＋-NTA resin affinity chromatography, the recombinant proreins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence （TRWLVYFSRPYLVAT） was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells （PBMC） up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE.展开更多
文摘Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.
基金Supported by Grants from the National Institutes of Health,No.AG029859 the National Center of the American Heart Association,No.0330335Nthe New Jersey Commission on Cancer Research(NJCCR703010)to Fan H
文摘AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.
文摘目的构建通用流感mRNA疫苗并全面评价其免疫保护效果。方法优化流感病毒株A/California/04/2009的血凝素(hemagglutinin,HA)、核蛋白(nucleoprotein,NP)和基质蛋白2胞外区(matrix protein 2 ectodomain,M2e)抗原序列,并将HA、NP和3个串联的M2e(3M2e)分别克隆至pcDNA3.1载体,通过线性化、体外转录、酶学法加帽、酶促加尾合成mRNA,命名为mRNA-HA、mRNA-NP和mRNA-3M2e。3种mRNA分别转染293T细胞后,通过免疫荧光实验鉴定蛋白的表达。利用脂质纳米颗粒分别包裹mRNA-HA、mRNA-NP和mRNA-3M2e并测量其粒径和电位。再将3种mRNA等体积混合制备成Comb-mRNA疫苗。将28只6周雌性Balb/c小鼠(体质量为18~22 g)按简单随机分组法分为2组:LNP组(n=14)和Comb-mRNA组(n=14)。通过血凝抑制(hemagglutination inhibition,HI)、微量中和(microneutralization,MN)实验评价流感mRNA疫苗诱导小鼠产生的血清抗体滴度;通过流式细胞术评价Comb-mRNA疫苗诱导的细胞免疫反应。采用5LD_(50)野生型H1N1流感病毒株感染小鼠评价Comb-mRNA疫苗的免疫保护效果。结果成功构建mRNA-HA、mRNA-NP和mRNA-3M2e,且3种mRNA均能在293T细胞表达。脂质纳米颗粒包裹mRNA平均粒径为(119.53±6.5)nm,平均电位为(-8.23±1.3)mV。与LNP组比较,Comb-mRNA组疫苗的HI几何平均滴度(geometric mean titer,GMT)达179.6,MN的GMT达201.6,同时诱导IFNγ+CD4+/CD8+T细胞比例升高。Comb-mRNA组在加强免疫后2周能够提供对5LD_(50)野生流感H1N1亚型病毒的保护。结论通用流感疫苗候选疫苗Comb-mRNA能够诱导小鼠免疫反应并保护小鼠免受病毒感染。
基金supported by National Nature Science Foundation of China (No.30070722).
文摘Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.
文摘Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （TS00） and full-length ectodomain （T1300） of TACE were amplified by RT-PCR, and the expres.sion plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant TS00 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that TS00 and T1300 were highly expressed in the form of inclusion body. After Ni^2＋-NTA resin affinity chromatography, the recombinant proreins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence （TRWLVYFSRPYLVAT） was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells （PBMC） up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE.
