Agro-industrial residues are primarily composed of complex polysaccharides that strengthen the microbial growth for the production of industrially important enzymes like cellulases. In the present study we aimed to ch...Agro-industrial residues are primarily composed of complex polysaccharides that strengthen the microbial growth for the production of industrially important enzymes like cellulases. In the present study we aimed to characterize the Exo 1, 4-β glucanase that was indigenously produced from Trichoderma viride MBL. T. viride MBL was cultured in the Solid-State medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 412 ± 12 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Exo 1, 4-β glucanase was 4.17-fold purified with specific activity of 642 U/mg in comparison to the crude extract. To confirm its purity and molecular weight, sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS- PAGE) was performed. The enzyme was shown to have a molecular weight of 60 kDa with an optimum pH and temperature of 5 and 50℃, respectively. Lineweaver-Burk reciprocal plot revealed that the kinetic constants Km and Vmax of purified Exo 1, 4-β glucanase were 76 μM and 240 U/mL.展开更多
核酸外切酶1(exonuclease 1,EXO1)已被证明在一些肿瘤中可作为具有临床意义的生物标志物,然而其在肺腺癌中的作用尚不清楚。为了研究EXO1在肺腺癌中的作用,我们首先利用癌症基因组图谱数据集(the cancer genome atlas,TCGA)对肺腺癌患...核酸外切酶1(exonuclease 1,EXO1)已被证明在一些肿瘤中可作为具有临床意义的生物标志物,然而其在肺腺癌中的作用尚不清楚。为了研究EXO1在肺腺癌中的作用,我们首先利用癌症基因组图谱数据集(the cancer genome atlas,TCGA)对肺腺癌患者各临床特征与EXO1表达量之间的关系进行了生物信息学分析,并通过蛋白质免疫印迹和CCK-8实验在肺腺癌细胞株中进行验证。结果显示,EXO1在肺腺癌中呈现高表达,并且其表达程度与肺腺癌的临床分期相关,我们推测EXO1是影响肺腺癌总体生存的独立危险因素;此外,通过基因富集分析确定了EXO1与DNA传感途径、碱基切除修复、DNA复制、细胞周期、P53信号通路和ERBB信号通路有关,这些途径在高EXO1表达的肺腺癌中高度富集。蛋白免疫印迹和CCK-8实验结果显示,EXO1在肺腺癌细胞中高表达,干扰EXO1表达水平可以抑制肺腺癌细胞的增殖能力。本研究证明了EXO1在肺腺癌的中高表达水平,它可以作为潜在的独立预后分子标志物,为肺腺癌患者的诊断治疗和预后评价提供新思路。展开更多
Exonuclease 1(EXO1)can catalyze nucleotide chain excision with its conserved N-terminal domain of 5′ to 3′ exonuclease activity,enabling it to influence diverse biological processes facing the challenges of genotoxi...Exonuclease 1(EXO1)can catalyze nucleotide chain excision with its conserved N-terminal domain of 5′ to 3′ exonuclease activity,enabling it to influence diverse biological processes facing the challenges of genotoxic environmental factors such as ionizing radiation.This nuclease activity enables EXO1 to maintain replication forks and telomeres length,to facilitate post-replication DNA repair and to process the end resection step of homologous recombination of DNA double-strand breaks-induced by ionizing radiation.When DNA replication is disrupted or blocked,EXO1 can cleave the broken DNA ends to form 3’ssDNA,leading to repair pathways activation.Excess EXO1-mediated nucleotide excision,however,can introduce an abundance of single-stranded DNA that can cause mutation and recombination via micro-homology-mediated end joining or single-strand annealing mechanisms,contributing to a loss of genetic information.EXO1 activity must therefore be carefully regulated within healthy cells.The mutations and dysregulations of EXO1 can increase the sensitivity of cells to radiation injury and risk of oncogenic transformation,limit the adoption of specific treatments in a range of human diseases.As such,EXO1 represents a promising target for the treatment and prevention of cancer.In the present review,we delineate the structural properties and functional characteristics of EXO1,discuss the relationship between this exonuclease and cancer susceptibility as well as the second cancers related to radiotherapy.展开更多
【目的】深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理,鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段,并分析其作用强度,明确蛋白间互作功能域。【方法】通过生物信息...【目的】深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理,鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段,并分析其作用强度,明确蛋白间互作功能域。【方法】通过生物信息学分析得到蛋白功能域,根据分析结果以典型的自交不亲和结球甘蓝E1为材料分别扩增SRK、ARC1和Exo70A1含不同功能域的截短体片段,利用分子克隆技术将SRK激酶域(SRKj)及其截短体SRKjΔ1—SRKjΔ4,Exo70A1全长及其截短体Exo70A1Δ1—Exo70A1Δ3的编码序列分别亚克隆至p GADT7(AD)质粒,将ARC1及其截短体ARC1Δ1—ARC1Δ8的编码序列分别亚克隆至载体p GBKT7(BD)质粒。用PEG/Li Ac法将获得的AD和BD重组质粒两两组合分别共转化到酵母AH109感受态中,观察融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT平板上的菌落生长情况和颜色变化情况,进一步测定其β-半乳糖苷酶活性。最后通过原核表达体外孵育检测蛋白质相互作用的方法对SRK-ARC1及ARC1-Exo70A1的相互作用进行验证。【结果】DNA测序和内切酶分析显示成功构建18个酵母双杂交表达载体,且无自激活能力。在SRK-ARC1的10个试验组合中,只有ARC1Δ4、ARC1Δ8、ARC1与SRKj组合的融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1。随着SRKj或ARC1截短体片段的延长,二者的β-半乳糖苷酶活性逐渐增加,其中,ARC1Δ4与SRKj组合的β-半乳糖苷酶活性最高(酶活为15.98)。在ARC1-Exo70A1 16个试验组合中,Exo70A1Δ3与ARC1Δ1Δ3都相互作用,其融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1。随着ARC1或Exo70A1截短体片段的延长,二者的β-半乳糖苷酶活性呈现先增加后降低的趋势,其中ARC1Δ2与Exo70A1Δ3组合的β-半乳糖苷酶活性最大(酶活性为25.07)。说明ARC1的N端和Exo70展开更多
The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, w...The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, we found that EXO7oA1 interacted with the GOLD domain of Patellin3 （PATL3）. The direct EXO7OA1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO7oA1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo7oa1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo7oe2, PRsec6 or exo84b mutants. These data suggested that EXO7oA1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicletethering/fusion. Both EXO7oA1 and PATL3 were shown to bind PI4P and PI（4,5）P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO7oA1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunitspecific function, and provided data and tools for further characterization of PATL family proteins.展开更多
In angiosperms,the process of pollination relies on species-specific interaction and signaling between the male(pollen)and female(pistil)counterparts where the interplay between several pollen and stigma proteins deci...In angiosperms,the process of pollination relies on species-specific interaction and signaling between the male(pollen)and female(pistil)counterparts where the interplay between several pollen and stigma proteins decides the fate of the pollen.In Brassicaceae,the dry stigmatic papillary cells control pollen germination by releasing resources only to compatible pollen thereby allowing pollen to hydrate and germinate.Despite the identification of a number of stigmatic proteins that facilitate pollination responses,the signaling mechanisms that regulate functions of these proteins have remained unknown.Here,we show that,in Arabidopsis,an extremely functionally redundant mitogen-activated protein kinase(MAPK)cascade is required for maintaining stigma receptivity to accept compatible pollen.Our genetic analyses demonstrate that in stigmas,five MAPK kinases(MKKs),MKK1/2/3/7/9 are required to transmit upstream signals to two MPKs,MPK3/4,to mediate compatible pollination.Compromised functions of these five MKKs in the quintuple mutant(mkk1/2/3RNAi/mkk7/9)phenocopied pollination defects observed in the m pk4RNAi/m pk3 double mutant.We further show that this MAPK nexus converges on Exo70A1,a previously identified stigma receptivity factor essential for pollination.Given that pollination is the crucial initial step during plant reproduction,understanding the mechanisms that govern successful pollination could lead to development of strategies to improve crop yield.展开更多
文摘Agro-industrial residues are primarily composed of complex polysaccharides that strengthen the microbial growth for the production of industrially important enzymes like cellulases. In the present study we aimed to characterize the Exo 1, 4-β glucanase that was indigenously produced from Trichoderma viride MBL. T. viride MBL was cultured in the Solid-State medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 412 ± 12 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Exo 1, 4-β glucanase was 4.17-fold purified with specific activity of 642 U/mg in comparison to the crude extract. To confirm its purity and molecular weight, sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS- PAGE) was performed. The enzyme was shown to have a molecular weight of 60 kDa with an optimum pH and temperature of 5 and 50℃, respectively. Lineweaver-Burk reciprocal plot revealed that the kinetic constants Km and Vmax of purified Exo 1, 4-β glucanase were 76 μM and 240 U/mL.
文摘核酸外切酶1(exonuclease 1,EXO1)已被证明在一些肿瘤中可作为具有临床意义的生物标志物,然而其在肺腺癌中的作用尚不清楚。为了研究EXO1在肺腺癌中的作用,我们首先利用癌症基因组图谱数据集(the cancer genome atlas,TCGA)对肺腺癌患者各临床特征与EXO1表达量之间的关系进行了生物信息学分析,并通过蛋白质免疫印迹和CCK-8实验在肺腺癌细胞株中进行验证。结果显示,EXO1在肺腺癌中呈现高表达,并且其表达程度与肺腺癌的临床分期相关,我们推测EXO1是影响肺腺癌总体生存的独立危险因素;此外,通过基因富集分析确定了EXO1与DNA传感途径、碱基切除修复、DNA复制、细胞周期、P53信号通路和ERBB信号通路有关,这些途径在高EXO1表达的肺腺癌中高度富集。蛋白免疫印迹和CCK-8实验结果显示,EXO1在肺腺癌细胞中高表达,干扰EXO1表达水平可以抑制肺腺癌细胞的增殖能力。本研究证明了EXO1在肺腺癌的中高表达水平,它可以作为潜在的独立预后分子标志物,为肺腺癌患者的诊断治疗和预后评价提供新思路。
基金supported by grants from the National Natural Science Foundation of China(Grant No.31870847).
文摘Exonuclease 1(EXO1)can catalyze nucleotide chain excision with its conserved N-terminal domain of 5′ to 3′ exonuclease activity,enabling it to influence diverse biological processes facing the challenges of genotoxic environmental factors such as ionizing radiation.This nuclease activity enables EXO1 to maintain replication forks and telomeres length,to facilitate post-replication DNA repair and to process the end resection step of homologous recombination of DNA double-strand breaks-induced by ionizing radiation.When DNA replication is disrupted or blocked,EXO1 can cleave the broken DNA ends to form 3’ssDNA,leading to repair pathways activation.Excess EXO1-mediated nucleotide excision,however,can introduce an abundance of single-stranded DNA that can cause mutation and recombination via micro-homology-mediated end joining or single-strand annealing mechanisms,contributing to a loss of genetic information.EXO1 activity must therefore be carefully regulated within healthy cells.The mutations and dysregulations of EXO1 can increase the sensitivity of cells to radiation injury and risk of oncogenic transformation,limit the adoption of specific treatments in a range of human diseases.As such,EXO1 represents a promising target for the treatment and prevention of cancer.In the present review,we delineate the structural properties and functional characteristics of EXO1,discuss the relationship between this exonuclease and cancer susceptibility as well as the second cancers related to radiotherapy.
文摘【目的】深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理,鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段,并分析其作用强度,明确蛋白间互作功能域。【方法】通过生物信息学分析得到蛋白功能域,根据分析结果以典型的自交不亲和结球甘蓝E1为材料分别扩增SRK、ARC1和Exo70A1含不同功能域的截短体片段,利用分子克隆技术将SRK激酶域(SRKj)及其截短体SRKjΔ1—SRKjΔ4,Exo70A1全长及其截短体Exo70A1Δ1—Exo70A1Δ3的编码序列分别亚克隆至p GADT7(AD)质粒,将ARC1及其截短体ARC1Δ1—ARC1Δ8的编码序列分别亚克隆至载体p GBKT7(BD)质粒。用PEG/Li Ac法将获得的AD和BD重组质粒两两组合分别共转化到酵母AH109感受态中,观察融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT平板上的菌落生长情况和颜色变化情况,进一步测定其β-半乳糖苷酶活性。最后通过原核表达体外孵育检测蛋白质相互作用的方法对SRK-ARC1及ARC1-Exo70A1的相互作用进行验证。【结果】DNA测序和内切酶分析显示成功构建18个酵母双杂交表达载体,且无自激活能力。在SRK-ARC1的10个试验组合中,只有ARC1Δ4、ARC1Δ8、ARC1与SRKj组合的融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1。随着SRKj或ARC1截短体片段的延长,二者的β-半乳糖苷酶活性逐渐增加,其中,ARC1Δ4与SRKj组合的β-半乳糖苷酶活性最高(酶活为15.98)。在ARC1-Exo70A1 16个试验组合中,Exo70A1Δ3与ARC1Δ1Δ3都相互作用,其融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 m M 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1。随着ARC1或Exo70A1截短体片段的延长,二者的β-半乳糖苷酶活性呈现先增加后降低的趋势,其中ARC1Δ2与Exo70A1Δ3组合的β-半乳糖苷酶活性最大(酶活性为25.07)。说明ARC1的N端和Exo70
基金supported by grant 31200236 from the National Natural Science Foundation of China(NSFC)Grants KYTZ201402 and KJQN201534 from the Fundamental Research Funds for the Central Universities in China+1 种基金Grant 130809001 from the Jiangsu University Superiority Discipline Construction ProjectThe laboratory of T.M.is cofunded by the Netherlands Organization for Scientific Research(NWO 867.15.020)
文摘The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid （Y2H） analysis, we found that EXO7oA1 interacted with the GOLD domain of Patellin3 （PATL3）. The direct EXO7OA1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO7oA1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo7oa1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo7oe2, PRsec6 or exo84b mutants. These data suggested that EXO7oA1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicletethering/fusion. Both EXO7oA1 and PATL3 were shown to bind PI4P and PI（4,5）P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO7oA1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunitspecific function, and provided data and tools for further characterization of PATL family proteins.
基金Natural Sciences and Engineering Research Council of Canada funding for M.A.S.
文摘In angiosperms,the process of pollination relies on species-specific interaction and signaling between the male(pollen)and female(pistil)counterparts where the interplay between several pollen and stigma proteins decides the fate of the pollen.In Brassicaceae,the dry stigmatic papillary cells control pollen germination by releasing resources only to compatible pollen thereby allowing pollen to hydrate and germinate.Despite the identification of a number of stigmatic proteins that facilitate pollination responses,the signaling mechanisms that regulate functions of these proteins have remained unknown.Here,we show that,in Arabidopsis,an extremely functionally redundant mitogen-activated protein kinase(MAPK)cascade is required for maintaining stigma receptivity to accept compatible pollen.Our genetic analyses demonstrate that in stigmas,five MAPK kinases(MKKs),MKK1/2/3/7/9 are required to transmit upstream signals to two MPKs,MPK3/4,to mediate compatible pollination.Compromised functions of these five MKKs in the quintuple mutant(mkk1/2/3RNAi/mkk7/9)phenocopied pollination defects observed in the m pk4RNAi/m pk3 double mutant.We further show that this MAPK nexus converges on Exo70A1,a previously identified stigma receptivity factor essential for pollination.Given that pollination is the crucial initial step during plant reproduction,understanding the mechanisms that govern successful pollination could lead to development of strategies to improve crop yield.
