非受體酪氨酸激酶c-Abl調(diào)節(jié)凋亡誘導(dǎo)因子AIF生物功能的研究
發(fā)布時間：2019-04-02 09:09
【摘要】：非受體酪氨酸激酶c-Ab1的在哺乳動物細胞內(nèi)的編碼基因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蛋白屬于非受體酪氨酸激酶Abl家族的成員,在哺乳動物細胞中能夠廣泛表達。生理狀態(tài)下c-Ab1蛋白存在于細胞質(zhì)和細胞核內(nèi),依靠其蛋白結(jié)構(gòu)域C端含有的核定位序列(NLS)和核輸出信號(NES)完成c-Ab1蛋白質(zhì)在細胞核與細胞質(zhì)之間的穿梭。細胞質(zhì)c-Abl的功能主要為抑制細胞凋亡、促進細胞增殖、誘導(dǎo)細胞轉(zhuǎn)化和腫瘤形成；細胞核c-Ab1在應(yīng)對一系列生理和藥物刺激時被激活,與細胞的凋亡密切相關(guān)。 凋亡誘導(dǎo)因子AIF是一種由16個外顯子基因編碼的保守的線粒體黃素蛋白。AIF蛋白N端含有線粒體定位序列MLS,C端含有氧化還原酶結(jié)構(gòu)域即FAD和NAD結(jié)合區(qū)包括兩個核定位序列NLS。AIF蛋白其結(jié)構(gòu)域中含有線粒體定位序列(MLS)和細胞核定位序列(NLS),在生理狀態(tài)下依賴它們來形使在線粒體和細胞核之間的穿梭功能。在正常生理狀態(tài)下,AIF位于線粒體內(nèi)外膜之間,可以籍其N末端FAD結(jié)合區(qū)和其氧化還原活性來行使氧化還原酶的功能,并具有維持線粒體的正常生存的作用。凋亡誘導(dǎo)因子刺激下,AIF水解掉N端50個氨基酸,從線粒體內(nèi)釋放進入細胞核,引起染色質(zhì)凝集,誘導(dǎo)細胞發(fā)生不依賴于caspase途徑的細胞凋亡 非受體酪氨酸激酶c-Abl和凋亡誘導(dǎo)因子AIF在細胞內(nèi)都能通過NLS進出細胞核,參與細胞的不同生理功能。本研究通過免疫印跡和Duolink檢測兩種蛋白在細胞內(nèi)存在的相互作用,c-Abl在細胞內(nèi)能夠磷酸化AIF,說明AIF可能是c-Ab1的潛在激酶底物,兩者可能是通過相互作用共同參與細胞的生理活性,并通過ROS刺激進一步證實兩者之間存在相互作用。本研究經(jīng)過免疫熒光技術(shù)進一步檢測出在STI571抑制c-Abl激酶活性狀態(tài)下,凋亡誘導(dǎo)因子AIF蛋白在細胞內(nèi)發(fā)生聚集,并從線粒體內(nèi)釋放出來,在c-Abl激酶活性受抑制的條件下,細胞內(nèi)線粒體的活性也受到影響。流式細胞儀檢測結(jié)果顯示,c-Abl激酶活性受抑制的條件下不能引起細胞發(fā)生凋亡現(xiàn)象,但是對STS引起的細胞凋亡作用有促進作用。 本研究首次發(fā)現(xiàn)細胞內(nèi)非受體酪氨酸激酶c-Abl受抑制的條件下細胞內(nèi)凋亡誘導(dǎo)因子AIF及線粒體發(fā)生的變化,并證實c-Abl與細胞內(nèi)AIF存在相互作用,可能是通過對AIF的作用影響細胞內(nèi)線粒體的活性。本研究為進一步探討Abl家族酪氨酸激酶對細胞內(nèi)凋亡誘導(dǎo)因子AIF的影響提供了新的依據(jù)。
[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.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346
【參考文獻】
相關(guān)期刊論文 前1條
1 王昌正;陳東立;曹誠;馬清鈞;;凋亡誘導(dǎo)因子與非受體酪氨酸激酶c-Abl的相互作用[J];生物技術(shù)通訊;2006年02期
，本文編號：2452439
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