Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability.Disordered apoptosis can cause a variety of diseases;therefore,sensing apoptosis can provide help in stud...Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability.Disordered apoptosis can cause a variety of diseases;therefore,sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development.It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis.Here,we present a genetically encoded switch-on m Neon Green2-based molecular biosensor.m Neon Green2 is the brightest monomeric green fluorescent protein.The substrate of caspase-3,DEVD amino acid residues,is inserted in it,while cyclized by insertion of Nostoc punctiforme Dna E intein to abolish the fluorescence(inactive state).Caspase-3-catalyzed cleavage of DEVD linearizes m Neon Green2 and rebuilds the natural barrel structure to restore the fluorescence(activated state).The characterization exhibited that the Caspase-3 biosensor has shortened response time,higher sensitivity,and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts.We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of He La and MCF-7 tumor cells and inhibition of Zika virus invasion.展开更多
目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过...目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过分子对接与生物传感技术揭示其与苦味受体的相互作用机制,实现苦味属性从传统经验判定到分子水平表征的跨越,为完善白术质量控制体系提供支撑。方法采用D101大孔吸附树脂结合紫外分光光度法,制备并筛选富含内酯类成分的白术组分;通过UPLC-MS/MS与分子对接技术,在分子水平解析白术组分与味觉受体的相互作用特征;采用生物传感器与UPLCMS/MS联用技术,实现白术组分苦味关键质量属性的精准辨识。结果成功制得白术不同极性组分11个,其中白术醇提物70%组分的总内酯含量最高,为后续性味研究的理想载体;分子对接实验显示白术组分与Ⅱ型味觉受体家族14号成员(taste receptor type 2 member 14,TAS2R14)的结合展现出明显优势,在分子层面揭示了白术组分具有强烈趋于苦味属性的作用特质;进一步构建苦味生物传感器,对白术组分的苦味属性进行表征,结果显示白术组分与TAS2R14的相互结合强度为49.0ng/L,属于强相互作用,表明以内酯类成分为主的白术组分具有显著的苦味属性,该结果与分子对接结果相互印证,增强了实验结论的可靠性;最后,联合UPLC-MS/MS技术,共辨识得到包括白术内酯Ⅱ在内的苦味关键质量属性13个,在物质基础层面表明白术组分与苦味受体TAS2R14的结合受关键质量属性驱动,为理解白术内酯类成分苦味特性提供直接证据。结论通过多技术融合的系统性研究,从分子相互作用到物质基础层面揭示了白术内酯类成分苦味属性的本质,所发现的苦味关键质量属性为建立基于苦味特征的白术质量控制方法奠定了理论基础,此外,提出的技术路线为中药苦味物质基础研究提供了新的方法学参考。展开更多
基金supported by the National Natural Science Foundation of China(21890743)National Key Research and Development Program of China(2017YFA0205500,2018YFA0902702)the Strategic Priority Research Program of the Chinese Academy of Sciences,China(XDB29050100)。
文摘Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability.Disordered apoptosis can cause a variety of diseases;therefore,sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development.It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis.Here,we present a genetically encoded switch-on m Neon Green2-based molecular biosensor.m Neon Green2 is the brightest monomeric green fluorescent protein.The substrate of caspase-3,DEVD amino acid residues,is inserted in it,while cyclized by insertion of Nostoc punctiforme Dna E intein to abolish the fluorescence(inactive state).Caspase-3-catalyzed cleavage of DEVD linearizes m Neon Green2 and rebuilds the natural barrel structure to restore the fluorescence(activated state).The characterization exhibited that the Caspase-3 biosensor has shortened response time,higher sensitivity,and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts.We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of He La and MCF-7 tumor cells and inhibition of Zika virus invasion.
文摘目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过分子对接与生物传感技术揭示其与苦味受体的相互作用机制,实现苦味属性从传统经验判定到分子水平表征的跨越,为完善白术质量控制体系提供支撑。方法采用D101大孔吸附树脂结合紫外分光光度法,制备并筛选富含内酯类成分的白术组分;通过UPLC-MS/MS与分子对接技术,在分子水平解析白术组分与味觉受体的相互作用特征;采用生物传感器与UPLCMS/MS联用技术,实现白术组分苦味关键质量属性的精准辨识。结果成功制得白术不同极性组分11个,其中白术醇提物70%组分的总内酯含量最高,为后续性味研究的理想载体;分子对接实验显示白术组分与Ⅱ型味觉受体家族14号成员(taste receptor type 2 member 14,TAS2R14)的结合展现出明显优势,在分子层面揭示了白术组分具有强烈趋于苦味属性的作用特质;进一步构建苦味生物传感器,对白术组分的苦味属性进行表征,结果显示白术组分与TAS2R14的相互结合强度为49.0ng/L,属于强相互作用,表明以内酯类成分为主的白术组分具有显著的苦味属性,该结果与分子对接结果相互印证,增强了实验结论的可靠性;最后,联合UPLC-MS/MS技术,共辨识得到包括白术内酯Ⅱ在内的苦味关键质量属性13个,在物质基础层面表明白术组分与苦味受体TAS2R14的结合受关键质量属性驱动,为理解白术内酯类成分苦味特性提供直接证据。结论通过多技术融合的系统性研究,从分子相互作用到物质基础层面揭示了白术内酯类成分苦味属性的本质,所发现的苦味关键质量属性为建立基于苦味特征的白术质量控制方法奠定了理论基础,此外,提出的技术路线为中药苦味物质基础研究提供了新的方法学参考。
