黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞...黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。展开更多
Objective Bouchardatine(1)is a β-indoloquinazoline alkaloid isolated from the plant Bouchardatia neurococca,acting as a modulator of adipogenesis and lipogenesis,and as an anticancer agent.The natural product functio...Objective Bouchardatine(1)is a β-indoloquinazoline alkaloid isolated from the plant Bouchardatia neurococca,acting as a modulator of adipogenesis and lipogenesis,and as an anticancer agent.The natural product functions as an activator of proteins adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)and sirtuin 1(SIRT1).We used molecular modeling to investigate the SIRT1-binding capacity of compound 1 and various structural analogues,such as orirenierine A(2)and orirenierine B(3)isolated from the medicinal plant Oricia renieri.Methods We investigated the binding to human SIRT1(hSIRT1)of 25 natural products including theβ-indoloquinazoline alkaloids 1−3 and analogues,in comparison with the reference product sirtinol(R and S isomers).A sirtinol binding model was elaborated starting from the closed and open state conformations of the catalytic domain of hSIRT1(PDB structures 4KXQ and 4IG9).For each compound bound to SIRT1,the empirical energy of interaction(ΔE)was calculated and compared to that of sirtinol.Results In our model,compound 1 was found to bind modestly to the sirtinol site of SIRT1.In contrast,the presence of a phenolic OH group at position 7 on the quinazolinone moiety conferred a much higher binding capacity.Compound 2 provided SIRT1 protein complexes as stable as those observed with sirtinol.The replacement of the hydroxy substituent(2)with a methoxy group(3)reduced the SIRT1 binding capacity.Other SIRT1-binding natural products were identified,such as the alkaloids orisuaveolines A and B.Structure-binding relationships were discussed.Conclusion The study underlines the capacity of β-indoloquinazoline alkaloids to interact with SIRT1.This deacetylase enzyme could represent a molecular target for the alkaloid 2.This compound merits further attention for the design of drugs active against SIRT1-dependent pathologies.展开更多
文摘黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。
文摘Objective Bouchardatine(1)is a β-indoloquinazoline alkaloid isolated from the plant Bouchardatia neurococca,acting as a modulator of adipogenesis and lipogenesis,and as an anticancer agent.The natural product functions as an activator of proteins adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)and sirtuin 1(SIRT1).We used molecular modeling to investigate the SIRT1-binding capacity of compound 1 and various structural analogues,such as orirenierine A(2)and orirenierine B(3)isolated from the medicinal plant Oricia renieri.Methods We investigated the binding to human SIRT1(hSIRT1)of 25 natural products including theβ-indoloquinazoline alkaloids 1−3 and analogues,in comparison with the reference product sirtinol(R and S isomers).A sirtinol binding model was elaborated starting from the closed and open state conformations of the catalytic domain of hSIRT1(PDB structures 4KXQ and 4IG9).For each compound bound to SIRT1,the empirical energy of interaction(ΔE)was calculated and compared to that of sirtinol.Results In our model,compound 1 was found to bind modestly to the sirtinol site of SIRT1.In contrast,the presence of a phenolic OH group at position 7 on the quinazolinone moiety conferred a much higher binding capacity.Compound 2 provided SIRT1 protein complexes as stable as those observed with sirtinol.The replacement of the hydroxy substituent(2)with a methoxy group(3)reduced the SIRT1 binding capacity.Other SIRT1-binding natural products were identified,such as the alkaloids orisuaveolines A and B.Structure-binding relationships were discussed.Conclusion The study underlines the capacity of β-indoloquinazoline alkaloids to interact with SIRT1.This deacetylase enzyme could represent a molecular target for the alkaloid 2.This compound merits further attention for the design of drugs active against SIRT1-dependent pathologies.
