【作者】 王艳；

【导师】 罗志军；

【作者基本信息】 南昌大学 ， 药理学， 2014， 硕士

【摘要】 背景和目的：5’单磷酸腺苷活化蛋白激酶（AMP-activated protein kinase，AMPK）是细胞的能量感受器，调节细胞能量代谢，在正常细胞和癌细胞中均发挥重要的生物功能，它的激活有助于纠正代谢紊乱，使其代谢趋向生理平衡。在细胞应急反应中，细胞感受到能量危机, ATP浓度下降，AMP浓度上升，细胞内AMP/ATP比例上升，AMPK被激活；在病理状态下，如代谢综合征、肿瘤等，常伴随能量代谢失调，AMPK活性下降，此时，药物激活AMPK可以使能量代谢恢复正常。因此，AMPK被视为治疗代谢性疾病与肿瘤的作用靶点。线粒体作为细胞的能量工厂，参与人体众多生理与病理过程，而AMPK对能量代谢的调节与线粒体的功能密不可分。越来越多研究表明，线粒体能影响AMPK的活性，同时AMPK也通过多方面对线粒体进行调节。但AMPK能否通过直接结合在细胞线粒体内而发挥调节细胞功能，目前尚无报道。本研究旨在解决这一问题并探明某些特定的功能是否归因于线粒体AMPK。方法:1. AMPK在线粒体的定位。采用免疫印迹和免疫荧技术检测AMPK在线粒体的定位。为检测AMPK定位于线粒体的决定片段，我们使HEK-293T细胞内表达AMPKα1亚基各片段，采用生化方法检测各片段是否定位于线粒体内。2.线粒体中AMPK的功能。为此目的，设计以慢病毒为载体，含有持续激活的AMPKα1亚基的重组病毒，且在该持续激活的亚基上连接靶向线粒体的肽段且在亚基氨基酸末端连接Flag抗原决定簇；将该重组病毒转染于肺癌细胞系-A549细胞中。随后研究线粒体AMPK对自噬的影响。结果：1.内源性AMPK包括α亚基和β亚基不仅分布于其它亚细胞结构，也分布在线粒体上。2. AMPKα1与线粒体的结合位点在其羧基末端的392-548氨基酸残基之间，该结合位点与亚基上的β亚基结合位点相重叠，由此表明α1亚基直接结合或者通过β亚基间接结合到线粒体上。3.线粒体靶向募集活性AMPK可以在无糖条件下增强自噬，表明线粒体AMPK在自噬中发挥重要作用。结论：1. AMPK存在于线粒体内且AMPKα1定位于线粒体的决定片段为其羧基末端：391-548氨基酸序列；2. AMPK在线粒体内对自噬发挥重要作用，表明在无糖时保护细胞；更多还原

【Abstract】 Background and objective：AMP-activated protein kinase (AMPK) serves as a fuel sensor that plays animportant role in regulating energy metabolism in all cells under physiological andpathological circumstances. AMPK is activated under stress, when cells sense energycrisis concurrent with decreases in ATP levels and increases in AMP or the ratio ofAMP to ATP. Such pathological conditions as metabolic syndrome and cancer areaccompanied by deregulated energy metabolism and suppression of AMPK, underwhich pharmacological activation of AMPK tends to tune it to a normal level. Assuch, AMPK emerges as a promising therapeutic target for these disorders. Theregulation of energy homeostasis by AMPK is closely related to the function ofmitochondrion, which acts as an energy plant and is involved in both physiologicaland pathological processes. Mounting evidence has demonstrated that mitochondriacan regulate AMPK activity and vice versa, AMPK also regulates mitochondrialfunction in various aspects. It has never been documented whether AMPK directlybinds to mitochondrion, thereby regulating cell function. The present study aims toaddress this question and ascribe specific cellular function to mitochondrial AMPK.Methods：1. Determine mitochondrial localization of AMPK. Biochemical fractionationfollowed by Western blot and immunofluorescence were used to localize AMPK tomitochondrion. To determin domains responsible for mitochondrial binding, differentmutants of AMPK α1subunit were expressed in HEK293T cells and their ability toassociate with mitochondria was examined by biochemical methods.2. Characterize function of mitochondrial AMPK. Toward this end,constitutively active mutant of AMPK α1subunit was engineered with amitochondrial targeting peptide followed by a Flag epitope at its aminoterminus inlentiviral vector and a lung adenocarcinoma cell line, A549, was infected with therecombinant virus expressing the AMPK mutant. The effect of mitochondrial AMPK on autophagy induction was then examined.Results：1. Endogenous AMPK including both α and β subunits is localized tomitochondria in addition to other subcellular compartments;2. The mitochondria binding site on AMPK α1subunit is localized to itscarboxyl terminal portion between aa392and548, overlapping with the bindingsite for the subunit, suggesting that the α1subunit directly associates withmitochondrion or indirectly through the subunit.3. Targeting active mutant of AMPK to mitochondria enhances autophagyinduced by glucose deprivation, suggesting that the mitochondrial AMPK plays arole in autophagy.Conclusion：1. AMPK exists in mitochondrion and The mitochondria binding site on AMPKα1subunit is localized to its carboxyl terminal portion between aa392and548.2. Mitochondriial AMPK plays an important role in regulating autophagy,sugestingAMPK can protect cells under no glucose.更多还原