【作者】 张媛；

【导师】 丁树哲； David A.Hood；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2013， 博士

【摘要】 线粒体是细胞内能量代谢“工厂”,是细胞代谢的重要器官。线粒体具有半自主性,即线粒体拥有独立的DNA,从而使得线粒体蛋白受到细胞核DNA与线粒体DNA双重调控。己知99%的线粒体蛋白由细胞核DNA编码调控,需要经过在细胞质中转录、翻译形成携带特殊信号序列的前体蛋白,这些前体蛋白在细胞质伴侣蛋白的协助下,通过位于线粒体膜上蛋白输入系统,输入到线粒体特定不同区域。输入至线粒体的前体蛋白在基质伴侣蛋白作用下形成成熟蛋白,最终在线粒体呼吸链或线粒体基质中发挥作用。由此看来,骨骼肌线粒体蛋白输入系统是影响骨骼肌线粒体生物发生的重要环节,是线粒体生物发生得以顺利进行的物质保证。线粒体蛋白输入机制(PIM, Protein Import Machinery)涉及线粒体蛋白输入系统相关组件及不同线粒体蛋白输入通路的功能。其中,线粒体蛋白输入系统的组件构成非常复杂,包括线粒体外膜转移酶复合物TOM、线粒体内膜转移酶复合物TIM、线粒体基质伴侣蛋白以及细胞质中协助前体蛋白输入的伴侣蛋白等诸多组件。目的：本研究主要以PGC-1α、Bax/Bak基因为切入点,根据PGC-1α、Bax/Bak基因的不同特征,通过分别深入研究PGC-1α、Bax/Bak基因与线粒体PIM组件蛋白表达以及线粒体PIM功能的关系,探讨不同应激对线粒体PIM组件蛋白表达及功能调节的分子作用机制,一方面首次关注PGC-1α、Bax/Bak基因是否对线粒体PIM具有调节作用,另一方面揭示调节线粒体PIM的分子机制。方法：1. PGC-1α基因对骨骼肌线粒体PIM作用机制研究1)细胞水平对PGC-1α基因的基础性研究：采用C2C12肌细胞模型,对C2C12细胞进行分化,生成可收缩的成熟肌管细胞。实验主要采用急性电刺激成熟肌细胞模型以及结合使用多种特异性抑制剂：如NAC (ROS↓)、BAPTA-AM (Ca2+↓)、Compound c (AMPK↓)、BIRB796(p38↓)等。观察急性电刺激条件下,不同分子信号通路对PGC-1α基因在转录水平以及翻译后蛋白水平的调控。实验Ⅰ：分为对照组、急性电刺激组以及急性电刺激恢复组。Western Blot方法检测PGC-1α基因相关分子信号通路蛋白表达,免疫荧光法检测急性电刺激对肌管细胞内PGC-1α蛋白迁移的影响。实验Ⅱ：分为对照组、对照组+抑制剂处理组、急性电刺激组、急性电刺激+抑制剂处理组。在肌细胞内利用细胞转染技术,转染预构建的i)含小鼠PGC-1α基因启动子全序列的荧光素质粒或ii)含PGC-1α蛋白DNA全序列的荧光素质粒,荧光素酶检测法分别检测不同分子信号通路对PGC-1α基因转录活性以及辅激活转录活性的影响。2)动物水平对PGC-1α基因与线粒体PIM关系进行研究：采用PGC-1α基因敲除鼠动物模型,对比研究PGC-1α野生型及基因敲除小鼠骨骼肌线粒体PIM组件蛋白表达以及PIM功能。实验分组：PGC-1α WT组与PGC-1α KO组。Western Blot方法检测不同线粒体PIM组件蛋白表达,放射性同位素35s标记示踪法检测线粒体PIM功能。2. Bax/Bak基因对骨骼肌线粒体PIM作用机制研究动物水平对Bax/Bak基因与线粒体PIM关系进行研究：采用Bax/Bak双基因敲除鼠动物模型,对比研究Bax/Bak基因的促细胞调亡特性及非促细胞凋亡特性,Bax/Bak野生型及双基因敲除小鼠骨骼肌线粒体PIM组件蛋白表达以及PIM功能。实验分为Bax/Bak WT组与Bax/Bak DKO组。Western Blot方法检测细胞素色C释放、线粒体融裂主要蛋白表达、线粒体PIM组件蛋白表达。JC-1线粒体跨膜电位指示剂检测线粒体膜电位。放射性同位素35S标记示踪法检测线粒体PIM功能。3.骨骼肌线粒体PIM运动适应性研究动物水平对骨骼肌线粒体PIM运动适应性分子机制进行研究：采用Bax/Bak双基因敲除鼠动物,结合6周自主性跑轮运动模型,对比研究Bax/Bak野生型及双基因敲除小鼠在运动训练条件下骨骼肌线粒体PIM组件蛋白表达及功能变化。实验分为Bax/Bak WT对照组、Bax/Bak WT运动组、Bax/Bak DKO对照组、Bax/BakDKO运动组。Western Blot方法检测不同线粒体PIM组件蛋白表达,放射性同位素35S标记示踪法检测线粒体PIM功能。结果：1.急性电刺激肌细胞通过细胞内ROS/Ca2+/AMPK/p38MAPK等分子／激酶信号通路,使PGC-1α基因转录活性与辅激活转录活性各显著增高52%、35%,为其参与调节线粒体PIM组件蛋白表达提供基础。2. PGC-1α基因缺失对骨骼肌线粒体PIM组件蛋白表达的影响呈现多样性,对骨骼肌线粒体PIM功能无显著影响。3. PGC-1α基因缺失造成的细胞质环境使骨骼肌线粒体OCT输入率显著降低44%-57%。4. Bax/Bak基因缺失使凋亡诱导因素刺激下的SS线粒体、IMF线粒体细胞色素C释放量各显著降低64%、70%,其在参与线粒体途径促细胞凋亡过程中发挥重要作用。5. Bax/Bak基因缺失导致线粒体基础态、态IV膜电位各显著降低20%和16%,并使线粒体融裂蛋白表达发生变化。6. Bax/Bak基因缺失使骨骼肌线粒体PIM组件蛋白Tom40、mtHsp60以及mtHsp70表达水平均显著降低38%至40%,却使细胞质内应激蛋白Hsp90、Bip表达水平异常显著增高,导致线粒体OCT、TomO蛋白输入率分别显著降低37%与41%,线粒体基质、外膜蛋白输入通路功能严重受损。7.运动训练上调线粒体PIM关键组件蛋白Tom40蛋白表达,优化细胞质应激蛋白表达水平,使受损的线粒体PIM功能恢复至正常水平。结论：1. Ca2+、ROS、AMPK、p38等细胞分子/激酶信号通路参与电刺激诱导的PGC-1α转录水平及翻译后蛋白水平基因调控,是PGC-1α在急性应激条件下作用于线粒体PIM组件基因的分子基础。2. PGC-la基因对骨骼肌线粒体PIM的调控并非作用于对TOM、TIM组件蛋白基因的调控,而通过对细胞质、线粒体基质相关参与内质网应激(ER stress)、未折叠蛋白反应(UPR, unfolded protein response)基因的调控。3)促细胞凋亡因子Bax/Bak基因除具有促细胞凋亡特性外,还具备许多潜在的非促细胞凋亡特性,具体表现在维持线粒体膜电位、参与调节线粒体融裂以及支持骨骼肌线粒体PIM等方面。4) Bax/Bak基因对骨骼肌线粒体基质蛋白(OCT)、线粒体外膜蛋白(Tom40)输入通路均具有重要作用,其主要通过对PIM组件蛋白表达、线粒体膜电位、PIM相关伴侣蛋白表达的影响得以实现。5)运动训练对Bax/Bak双基因敲除鼠受损线粒体蛋白输入功能具有正向调节的作用,这一作用伴随线粒体PIM组件蛋白表达运动适应性上调、细胞质中应激蛋白表达运动适应性优化。提示：骨骼肌线粒体PIM具有运动可塑性,且线粒体PIM与细胞质、内质网应激(ER stress)、未折叠蛋白反应(UPR, unfolded protein response)、线粒体应激等生理过程密切相关。更多还原

【Abstract】 Mitochondria are the "power house" within the cell and are the key organelle to cell metabolism. Mitochondrial proteins are encoded by nuclear and mitochondrial DNA. As we known,99%of mitochondrial proteins are encoded by nuclear DNA, those proteins have to be transcribed and translated, then formed as precursor proteins, carried specific signal sequences, in cytosol. With the help of cytosolic chaperone proteins, precursor proteins are imported to different compartments of mitochondria through mitochondrial protein import system on mitochondrial membrane. Once imported into mitochondria, mitochondrial enzyme will cut off the specific signal sequences of precursor proteins and form to mature proteins that participate in mitochondrial respiratory chain formation or other mitochondrial biogenesis process. Therefore, mitochondrial protein import machinery (PIM) is an essential step of regulating mitochondrial biogenesis. PIM concerns about hundreds of components of mitochondrial protein import and the function of different import pathways and it is composed of mitochondrial outer membrane translocases complex (TOM)、mitochondrial inner membrane translocases complex (TIM)、matrix chaperone proteins and cytosolic chaperone proteins.Purpose:PGC-1α and Bax/Bak were used as entry point in this research, based on their own features, we investigated the mechanisms of regulating mitochondrial protein import components expression and function in skeletal muscle in response to different stress through determining the role of PGC-1α and Bax/Bak in mitochondrial PIM components expression and PIM function, which is the first time to focus on whether PGC-1α or Bax/Bak play a role in mitochondrial PIM and reveal the regulation molecular mechanisms of mitochondrial PIM.Methods:1. The role of PGC-1α in mitochondrial PIM in skeletal muscle1) Cell culture research:C2C12myotubes were used to subject2hours acute contractile activity, with or without the treatment of specific inhibitors, such as NAC (ROS↓)、 BAPTA-AM (Ca2+↓)、Compound c (AMPK↓)、BIRB796(p38↓).Investigate the involvement of multiple signaling pathways in PGC-1αgene regulation in response to acute stimulation. Experiment Ⅰ:Group:Control, Stimulation and Stimulation+Recovery. Western Blot method was used to examine protein expression involved in PGC-1αrelated multiple signaling pathways and immunoflurencense method was used to determine the role of contractile activity on PGC-1α protein translocation within the myotube.Experiment Ⅱ:Group:Control, Control+Inhibitor, Stimulation, Stimulation+Inhibitor. Myoblasts were transfected with ⅰ) the full length of mouse PGC-1α promoter sequence fused with a luciferase vector, or ⅱ) the full length of PGC-1α protein DNA sequence fused with a luciferase vector. Luciferase assay was used to look at the role of multiple signaling pathways on the activity of PGC-1α transcription and coactivation.2) Animal research:PGC-la genetic mice were used in this study. Mitochondrial PIM components expression and function were examined in both PGC-la WT and PGC-la KO mice skeletal muscle. Group:PGC-1α WT and PGC-1α KO. Western Blot method was used to investigate mitochondrial PIM components expression, radiolabeled protein was used to look at mitochondrial PIM function.2. The role of Bax/Bak in mitochondrial PIM in skeletal muscleAnimal research:Bax/Bak double knock out mice were used in this study. Bax/Bak apoptotic role, Bax/Bak non-apoptotic roles, mitochondrial PIM components expression and PIM function were examined in both Bax/Bak WT and Bax/Bak DKO mice skeletal muscle. Group:Bax/Bak WT and Bax/Bak DKO. Western Blot method was used to determine the release of cytochrome c, mitochondrial dynamic proteins expression and mitochondrial PIM components expression. JC-1dye was used to look at mitochondrial membrane potential. Radiolabeled protein with35S was used to look at mitochondrial PIM function.3. Exercise adaptation of mitochondrial PIM in skeletal muscleAnimal research:Bax/Bak double knock out mice were used in this study, combined with6-week voluntary running wheel. Mitochondrial PIM components expression and function were investigated between Bax/Bak WT and Bax/Bak DKO mice in the absence or presence of exercise training. Group:Bax/Bak WT Control, Bax/Bak DKO Control, Bax/Bak WT training, Bax/Bak DKO training. Western Blot method was used to investigate mitochondrial PIM components expression. Synthesized test protein radiolablled with35S was used to look at mitochondrial PIM function.Results:1. The activity of PGC-la transcription and coactivation were significantly increased by52%and35%respectively, in response to acute contractile activity through cellular ROS/Ca2+/AMPK/p38MAPK molecular signaling pathways.2. The absence of PGC-la has different effects on mitochondrial PIM components expression, and has no influence on PIM function in skeletal muscle.3. Cytosol with the absence of PGC-la cause44%to57%significantly decreased in mitochondrial OCT import in skeletal muscle.4. Cytochrome c release from SS and IMF mitochondria were both significantly reduced by64%and70%, respectively, in Bax/Bak DKO mice under stimuli treatment, which suggest Bax/Bak plays an important role in mitochondrial induced cell apoptosis.5. Basal and state IV mitochondrial membrane potential were both significantly reduced by20%and16%with the absence of Bax and Bak, which associated with changed mitochondrial dynamic proteins expression.6. Mitochondrial PIM components Tom40、mtHsp60and mtHsp70expression were all significantly reduced from38%to40%in Bax/Bak DKO mice, whereas cytosolic stress proteins Hsp90and Bip displayed abnormal high level, this resulted in the import of OCT and Tom40protein were both significantly decreased by37%and41%, mitochondrial matrix and outer membrane protein import pathway were both impaired.7. Exercise training up-regulated the key mitochondrial PIM components expression, Tom40and optimized cytosolic stress proteins expression in skeletal muscle, which recovery the defect mitochondrial PIM function.Conclusions1. Multiple cellular signaling pathways:Ca2+、ROS、AMPK、p38were involved in the regulation of acute contractile activity-induced PGC-1αgene expression, both in transcriptional and post-translation level.2. The role of PGC-la in mitochondrial PIM in skeletal muscle rather through regulating gene expression that involved in ER stress and UPR, but not related to regulating TOM、TIM components expression.3. Bax/Bak has both apoptotic and non-apoptotic functions, the non-apoptotic functions manifested in maintaining mitochondrial membrane potential, regulating mitochondrial dynamic process and support mitochondrial PIM in skeletal muscle.4. Bax/Bak plays a vital role in the function of mitochondrial matrix and outer membrane protein import pathways, via regulating PIM components expression, mitochondrial membrane potential and PIM related chaperone proteins.5. Impaired function of mitochondrial protein import in Bax/Bak DKO mice was recovery by exercise training, which was associated with up-regulation of mitochondrial PIM components expression and optimized cytosolic stress proteins expression. This suggested exercise adaptation of mitochondrial PIM may relate to ER stress, UPR or mitochondrial stress.更多还原