非受体酪氨酸激酶c-Abl调节凋亡诱导因子AIF生物功能的研究
发布时间：2019-04-02 09:09
【摘要】：非受体酪氨酸激酶c-Ab1的在哺乳动物细胞内的编码基因c-abl是v-abl的同源基因,v-ab1是Abelson鼠白血病病毒原癌基因。c-Ab1蛋白属于非受体酪氨酸激酶Abl家族的成员,在哺乳动物细胞中能够广泛表达。生理状态下c-Ab1蛋白存在于细胞质和细胞核内,依靠其蛋白结构域C端含有的核定位序列(NLS)和核输出信号(NES)完成c-Ab1蛋白质在细胞核与细胞质之间的穿梭。细胞质c-Abl的功能主要为抑制细胞凋亡、促进细胞增殖、诱导细胞转化和肿瘤形成；细胞核c-Ab1在应对一系列生理和药物刺激时被激活,与细胞的凋亡密切相关。 凋亡诱导因子AIF是一种由16个外显子基因编码的保守的线粒体黄素蛋白。AIF蛋白N端含有线粒体定位序列MLS,C端含有氧化还原酶结构域即FAD和NAD结合区包括两个核定位序列NLS。AIF蛋白其结构域中含有线粒体定位序列(MLS)和细胞核定位序列(NLS),在生理状态下依赖它们来形使在线粒体和细胞核之间的穿梭功能。在正常生理状态下,AIF位于线粒体内外膜之间,可以籍其N末端FAD结合区和其氧化还原活性来行使氧化还原酶的功能,并具有维持线粒体的正常生存的作用。凋亡诱导因子刺激下,AIF水解掉N端50个氨基酸,从线粒体内释放进入细胞核,引起染色质凝集,诱导细胞发生不依赖于caspase途径的细胞凋亡 非受体酪氨酸激酶c-Abl和凋亡诱导因子AIF在细胞内都能通过NLS进出细胞核,参与细胞的不同生理功能。本研究通过免疫印迹和Duolink检测两种蛋白在细胞内存在的相互作用,c-Abl在细胞内能够磷酸化AIF,说明AIF可能是c-Ab1的潜在激酶底物,两者可能是通过相互作用共同参与细胞的生理活性,并通过ROS刺激进一步证实两者之间存在相互作用。本研究经过免疫荧光技术进一步检测出在STI571抑制c-Abl激酶活性状态下,凋亡诱导因子AIF蛋白在细胞内发生聚集,并从线粒体内释放出来,在c-Abl激酶活性受抑制的条件下,细胞内线粒体的活性也受到影响。流式细胞仪检测结果显示,c-Abl激酶活性受抑制的条件下不能引起细胞发生凋亡现象,但是对STS引起的细胞凋亡作用有促进作用。 本研究首次发现细胞内非受体酪氨酸激酶c-Abl受抑制的条件下细胞内凋亡诱导因子AIF及线粒体发生的变化,并证实c-Abl与细胞内AIF存在相互作用,可能是通过对AIF的作用影响细胞内线粒体的活性。本研究为进一步探讨Abl家族酪氨酸激酶对细胞内凋亡诱导因子AIF的影响提供了新的依据。
[Abstract]:The coding gene c-abl of non-receptor tyrosine kinase c-Ab1 in mammalian cells is a homologue of v-abl and v-ab1 is a proto-oncogene of Abelson mouse leukemia virus. The c-Ab1 protein belongs to the Abl family of non-receptor tyrosine kinase. It can be widely expressed in mammalian cells. In physiological condition, c-Ab1 protein exists in cytoplasm and nucleus. The nuclear localization sequence (NLS) and nuclear output signal (NES) (NES) in the C-terminal of c-Ab1 protein domain are used to complete the shuttle of c-Ab1 protein between nucleus and cytoplasm. The function of cytoplasmic c-Abl is to inhibit cell apoptosis, promote cell proliferation, induce cell transformation and tumor formation, and nuclear c-Ab1 is activated in response to a series of physiological and drug stimuli, which is closely related to cell apoptosis. Apoptosis inducer AIF is a conserved mitochondrial flavin encoded by 16 exon genes. The N terminal of AIF protein contains the mitochondrial localization sequence MLS,. The C terminal contains the FAD and NAD binding domains, including two nuclear localization sequences, NLS.AIF protein, which contain mitochondrial localization sequence (MLS) and nuclear localization sequence (NLS),. Depending on them in the physiological state to shape the shuttle function between mitochondria and nuclei. Under normal physiological condition, AIF is located between mitochondria in vivo and outer membrane. It can perform the function of oxidoreductase by means of its N-terminal FAD binding region and its redox activity, and can maintain the normal survival of mitochondria. Under the stimulation of apoptosis inducer, AIF hydrolyzed 50 amino acids at the N-terminal and released into the nucleus from mitochondria, causing chromatin agglutination. The induction of apoptosis by non-receptor tyrosine kinase c-Abl and apoptosis-inducing factor AIF, which are independent of caspase pathway, can enter and exit the nucleus through NLS in cells, and participate in different physiological functions of cells. In this study, immunoblotting and Duolink were used to detect the interaction between the two proteins in the cell. The ability of c-Abl to phosphorylate AIF, in cells indicates that AIF may be a potential kinase substrate of c-Ab1. Both of them may participate in the physiological activity of cells through interaction, and further confirm the interaction between them through ROS stimulation. In this study, immunofluorescence technique was used to detect the aggregation of apoptosis inducible factor AIF protein in the cells under the condition of STI571 inhibiting c-Abl kinase activity, and released from mitochondria, under the condition that the activity of c-Abl kinase was inhibited, and the activity of c-Abl kinase was inhibited under the condition of inhibition of c-Abl kinase activity. The activity of mitochondria is also affected. The results of flow cytometry showed that the inhibition of c-Abl kinase activity could not induce apoptosis, but could promote the apoptosis induced by STS. In this study, we found for the first time the changes of intracellular apoptosis-inducing factor AIF and mitochondria under the condition of inhibition of non-receptor tyrosine kinase c-Abl, and confirmed the interaction between c-Abl and intracellular AIF. It is possible that the activity of mitochondria in the cell is affected by the action of AIF. This study provides a new basis for further exploring the effect of Abl family tyrosine kinase on intracellular apoptosis-inducing factor AIF.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
本文编号：2452439
[Abstract]:The coding gene c-abl of non-receptor tyrosine kinase c-Ab1 in mammalian cells is a homologue of v-abl and v-ab1 is a proto-oncogene of Abelson mouse leukemia virus. The c-Ab1 protein belongs to the Abl family of non-receptor tyrosine kinase. It can be widely expressed in mammalian cells. In physiological condition, c-Ab1 protein exists in cytoplasm and nucleus. The nuclear localization sequence (NLS) and nuclear output signal (NES) (NES) in the C-terminal of c-Ab1 protein domain are used to complete the shuttle of c-Ab1 protein between nucleus and cytoplasm. The function of cytoplasmic c-Abl is to inhibit cell apoptosis, promote cell proliferation, induce cell transformation and tumor formation, and nuclear c-Ab1 is activated in response to a series of physiological and drug stimuli, which is closely related to cell apoptosis. Apoptosis inducer AIF is a conserved mitochondrial flavin encoded by 16 exon genes. The N terminal of AIF protein contains the mitochondrial localization sequence MLS,. The C terminal contains the FAD and NAD binding domains, including two nuclear localization sequences, NLS.AIF protein, which contain mitochondrial localization sequence (MLS) and nuclear localization sequence (NLS),. Depending on them in the physiological state to shape the shuttle function between mitochondria and nuclei. Under normal physiological condition, AIF is located between mitochondria in vivo and outer membrane. It can perform the function of oxidoreductase by means of its N-terminal FAD binding region and its redox activity, and can maintain the normal survival of mitochondria. Under the stimulation of apoptosis inducer, AIF hydrolyzed 50 amino acids at the N-terminal and released into the nucleus from mitochondria, causing chromatin agglutination. The induction of apoptosis by non-receptor tyrosine kinase c-Abl and apoptosis-inducing factor AIF, which are independent of caspase pathway, can enter and exit the nucleus through NLS in cells, and participate in different physiological functions of cells. In this study, immunoblotting and Duolink were used to detect the interaction between the two proteins in the cell. The ability of c-Abl to phosphorylate AIF, in cells indicates that AIF may be a potential kinase substrate of c-Ab1. Both of them may participate in the physiological activity of cells through interaction, and further confirm the interaction between them through ROS stimulation. In this study, immunofluorescence technique was used to detect the aggregation of apoptosis inducible factor AIF protein in the cells under the condition of STI571 inhibiting c-Abl kinase activity, and released from mitochondria, under the condition that the activity of c-Abl kinase was inhibited, and the activity of c-Abl kinase was inhibited under the condition of inhibition of c-Abl kinase activity. The activity of mitochondria is also affected. The results of flow cytometry showed that the inhibition of c-Abl kinase activity could not induce apoptosis, but could promote the apoptosis induced by STS. In this study, we found for the first time the changes of intracellular apoptosis-inducing factor AIF and mitochondria under the condition of inhibition of non-receptor tyrosine kinase c-Abl, and confirmed the interaction between c-Abl and intracellular AIF. It is possible that the activity of mitochondria in the cell is affected by the action of AIF. This study provides a new basis for further exploring the effect of Abl family tyrosine kinase on intracellular apoptosis-inducing factor AIF.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 王昌正;陈东立;曹诚;马清钧;;凋亡诱导因子与非受体酪氨酸激酶c-Abl的相互作用[J];生物技术通讯;2006年02期
，本文编号：2452439
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