CN108610410A - 一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法 - Google Patents
一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法 Download PDFInfo
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Abstract
一种Keap1‑Nrf2‑ARE通路抑制因子的快速筛选方法,所述的快速筛选方法是:修饰Keap1的半胱氨酸残基,使Nrf2在细胞核内积聚,并且表达Nrf2的靶基因。在正常条件下,Nrf2与Keap1结合,存在于细胞之中,发生氧化应激时,Keap1的半胱氨酸残基被修饰,改变构象导致Nrf2释放出来,进入细胞核中,与ARE结合后,促进靶基因的表达;诱导剂无法使Keap1‑Nrf2解离,并且Nrf2可以经泛素化后降解;Keap1通过其BTB区结合Cul3、Kelch区结合Nrf2,将Nrf2连接到E3复合体,使泛素从E3转移到Nrf2的赖氨酸残基,即位于ETGE基序、DLG基序之间,泛素化的Nrf2被迅速降解。发生氧化应激时,Keap1特定的半胱氨酸残基被修饰,导致构象的改变,DLG基序与Keap1的亲和力减弱而分离,即所谓的hinge and latch模型,从而免于泛素化降解。
Description
技术领域
本发明涉及的是一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法。
背景技术
癌症发生以后Keap1-Nrf2通路的高表达或过度积累会降低化疗或放疗引起的过度氧化应激对肿瘤细胞的伤害,帮助其抵抗化学药物或放射治疗,有利于肿瘤细胞的存活并促进其生长。此外,在肿瘤发生之后或在恶性肿瘤中Nrf2的过度激活会引起肿瘤细胞对化疗或放射疗法的抵抗和耐受性、导致患者预后不良。由此看来,肿瘤一旦发生,Nrf2因子在细胞内积累,不但不利于肿瘤的预防,反而会增强肿瘤细胞的耐药性并促进其生长,不利于肿瘤的治疗和患者的康复。因此,Keap1-Nrf2-ARE通路抑制因子的快速筛选方法具有重要意义。
发明内容
本发明的目的在于克服现有技术存在的不足,而提供一种可以快速、高效的筛选出具有Nrf2抑制作用的活性因子的、通过Nrf2抑制因子的发现为肿瘤的预防和治疗提供新方法的Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法。
本发明的目的是通过如下技术方案来完成的,一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法,所述的快速筛选方法是:修饰Keap1的半胱氨酸残基,使Nrf2在细胞核内积聚,并且表达Nrf2的靶基因。
作为优选:在正常条件下,Nrf2与Keap1结合,存在于细胞之中,发生氧化应激时,Keap1的半胱氨酸残基被修饰,改变构象导致Nrf2释放出来,进入细胞核中,与ARE结合后,促进靶基因的表达。
作为优选:诱导剂无法使Keap1-Nrf2解离,并且Nrf2可以经泛素化后降解;Keap1通过其BTB区结合Cul3、Kelch区结合Nrf2,将Nrf2连接到E3复合体,使泛素从E3转移到Nrf2的赖氨酸残基,即位于ETGE基序、DLG基序之间,泛素化的Nrf2被迅速降解。发生氧化应激时,Keap1特定的半胱氨酸残基被修饰,导致构象的改变,DLG基序与Keap1的亲和力减弱而分离,即所谓的hinge and latch模型,从而免于泛素化降解;另外,多种蛋白激酶,例如MAPKs、PKC、PI3K等,可通过诱导Nrf2的磷酸化参与对Nrf2转录活性的调节。
本发明可以快速、高效的筛选出具有Nrf2抑制作用的活性因子的、通过Nrf2抑制因子的发现为肿瘤的预防和治疗提供新方法。
具体实施方式
下面将结合具体实施例对本发明作详细的介绍:外来物质(致癌物、药物及环境污染物等)在体内一般经历两相代谢过程,一相代谢过程主要是官能团反应,在P450酶性的参与下,机体对药物分子进行氧化、还原、羟化、水解等,使大部分外来物质失活;二相代谢过程主要是呃合反应,在有关酶的催化下,将内源性极性小分子物质,如葡萄糖醛酸、谷胱甘肽等,经共价键结合外来物或一相代谢活化物的分子上,使之失活解毒;
致癌物质在体内的激活主要发生在一相代谢阶段,而二相代谢可以通过结合反应来减少它们对机体的毒害;二相代谢过程是在一系列酶的参与下完成的,它们能清除体内多种致癌物质及其它毒性或氧化性物质,从而消除这些物质对DNA及生物功能蛋白的破坏,以维持机体内环境的稳定,起到抗癌、抗氧化、抗衰老的作用,一般把这种酶统称为药物代谢二相酶,因此诱导这些酶的表达是预防癌症的重要措施,而二相酶的表达主要是通过Keap1-Nrf2-ARE 通路。
Nrf2是一种66000分子量的蛋白质,共有一个基本的亮氨酸结构域,几乎在各种细胞内都可表达,它可以与DNA的NF-EZ区结合,在造血细胞中,NF-EZ区参与调节球蛋白基因的表达。
转录因子Nrf2首先和肌腱纤维瘤蛋白以异二聚体的形式结合,然后该异二聚体再与ARE结合,从而启动二相酶基因的转录。
一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法,所述的快速筛选方法是:修饰Keap1的半胱氨酸残基,使Nrf2在细胞核内积聚,并且表达Nrf2的靶基因。
本发明在正常条件下,Nrf2与Keap1结合,存在于细胞之中,发生氧化应激时,Keap1的半胱氨酸残基被修饰,改变构象导致Nrf2释放出来,进入细胞核中,与ARE结合后,促进靶基因的表达;Nrf2高表达对肿瘤细胞有益。
诱导剂无法使Keap1-Nrf2解离,并且Nrf2可以经泛素化后降解;Keap1通过其BTB区结合Cul3、Kelch区结合Nrf2,将Nrf2连接到E3复合体,使泛素从E3转移到Nrf2的赖氨酸残基,即位于ETGE基序、DLG基序之间,泛素化的Nrf2被迅速降解。发生氧化应激时,Keap1特定的半胱氨酸残基被修饰,导致构象的改变,DLG基序与Keap1的亲和力减弱而分离,即所谓的hinge and latch模型,从而免于泛素化降解;另外,多种蛋白激酶,例如MAPKs、PKC、PI3K等,可通过诱导Nrf2的磷酸化参与对Nrf2转录活性的调节。
Claims (3)
1.一种Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法,其特征在于;所述的快速筛选方法是:修饰Keap1的半胱氨酸残基,使Nrf2在细胞核内积聚,并且表达Nrf2的靶基因。
2.根据权利要求1所述的Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法,其特征在于:在正常条件下,Nrf2与Keap1结合,存在于细胞之中,发生氧化应激时,Keap1的半胱氨酸残基被修饰,改变构象导致Nrf2释放出来,进入细胞核中,与ARE结合后,促进靶基因的表达。
3.根据权利要求1所述的Keap1-Nrf2-ARE 通路抑制因子的快速筛选方法,其特征在于:诱导剂无法使Keap1-Nrf2解离,并且Nrf2可以经泛素化后降解;Keap1通过其BTB区结合Cul3、Kelch区结合Nrf2,将Nrf2连接到E3复合体,使泛素从E3转移到Nrf2的赖氨酸残基,即位于ETGE基序、DLG基序之间,泛素化的Nrf2被迅速降解;发生氧化应激时,Keap1特定的半胱氨酸残基被修饰,导致构象的改变,DLG基序与Keap1的亲和力减弱而分离,即所谓的hinge and latch模型,从而免于泛素化降解;另外,多种蛋白激酶,例如MAPKs、PKC、PI3K等,可通过诱导Nrf2的磷酸化参与对Nrf2转录活性的调节。
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