Retrograde Signaling in Alcohol-Induced Mitochondrial Stress and Biogenesis.

酒精诱导的线粒体应激和生物发生中的逆行信号传导。

• 批准号: 7522592
• 负责人: NEIL KAPLOWITZ
• 金额: $ 19.36万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7522592
• 关键词: Acute; Agreement; Alcohol-Induced Disorders; Alcoholic Liver Diseases; Alcohols; Antimycin A; Biogenesis; Bioinformatics; Biological Assay; CREB1 gene; Cell Nucleus; Cells; Chronic; Cultured Cells; Cyclic AMP-Responsive DNA-Binding Protein; Cytosol; Data; Density Gradient Centrifugation; Endoplasmic Reticulum; Evolution; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Hepatocyte; Homeostasis; Image; Impairment; Injury; Laboratories; Liver; Mediating; Mitochondria; Mitochondrial Matrix; Modeling; Modification; Molecular Chaperones; Mus; Mutation; Nuclear; Nuclear Translocation; Organelles; Oxidative Stress; Pathogenesis; Pathway interactions; Pattern; Physiology; Play; Polymerase Chain Reaction; Predisposition; Process; Production; Protein Family; Proteins; Pyrazoles; Rattus; Relative (related person); Resolution; Role; Rotenone; Signal Pathway; Signal Transduction; Small Interfering RNA; Stress; Testing; Time; Transcriptional Activation; Transducers; Up-Regulation; Western Blotting; Work; alcohol exposure; alcohol response; biological adaptation to stress; concept; feeding; in vivo; in vivo Model; novel; promoter; protein misfolding; pyrazole; response; sensor; theories; transcription factor

DESCRIPTION (provided by applicant): There is widespread agreement that chronic alcohol exposure leads to mitochondrial damage. An unexplored aspect of mitochondrial homeostasis is the mitochondrial stress response (MSR) which regulates matrix chaperone production (e.g., Hsp60 and 10) and mitochondrial biogenesis in response to stress to mitochondria (e.g., protein malfolding or oxidative modifications) as would occur from alcohol. The MSR and biogenesis responses are mediated mainly by up-regulation of nuclear genes. We have begun to explore the upstream sensor and trigger of these responses, referred to as mitochondrial retrograde signaling and our Preliminary Results have identified a key transcriptional co-activator (TORC3) which resides in the mitochondrial matrix and appears to translocate to the nucleus after mitochondrial damage. Therefore, the aims of the current application are to: 1) Confirm localization of TORC3 in mitochondria and compare with localization of TORC2. TORC 2 and 3 are two transcriptional co-activators in liver which up-regulate Hsp60 and PGC-1a. The hypothesis that there is separate compartmentation of TORC2 (cytosol) and TORC3 (mitochondria matrix) will be carefully tested defined using differential or density gradient centrifugation, mitoplasts and high resolution confocal imaging: 2) Examine the role of TORC3 in MSR and mitochondrial biogenesis in cell culture models (HepG2 cells and primary mouse hepatocytes) of mitochondrial damage using rotenone, antimycin A, or expression of Cyp2e1 in mitochondria with the goals of (a) characterizing the detailed time course of TORC3 release from mitochondria and nuclear translocation in relation to MSR and biogenesis gene expression in response to mitochondrial oxidative stress (b) comparing nuclear translocation of TORC2 versus TORC3 to Hsp60 promoter and the effect of silencing TORC2 and or TORC3 on MSR and mitochondrial biogenesis as well as susceptibility to lethal mitochondrial injury; (c) determining the role of CHOP and concomitant ER stress in relation to TORC2 and TORC3 on mitochondrial stress and biogenesis responses; these studies will utilize real-time PCR, Western blots, ChIP assays and siRNA; 3) Define the effect of pyrazole-induced Cyp2e1 on MSR and mitochondrial biogenesis in vivo; preliminary results confirm pyrazole induces mitochondrial and ER Cyp2e1 and this causes MSR; therefore, pyrazole treatment may provide a convenient in vivo model for selective mitochondrial damage; 4) Describe the expression pattern of MSR and mitochondrial biogenesis genes in chronic intragastric alcohol fed mice. The results of this application should convincingly characterize a novel direct retrograde signaling pathway from mitochondria to nucleus involving TORC3 transcription co-activator, define its role relative to other signaling pathways, and begin to establish its relevance to alcohol injury in vivo.

描述(由申请人提供)：人们普遍认为长期接触酒精会导致线粒体损伤。线粒体动态平衡的一个未被研究的方面是线粒体应激反应(MSR)，它调节基质伴侣的产生(例如Hsp60和10)和线粒体的生物发生，以响应对线粒体的压力(例如蛋白质错误折叠或氧化修饰)，就像酒精所发生的那样。MSR和生物发生反应主要通过核基因的上调来调节。我们已经开始探索这些反应的上游传感器和触发，称为线粒体逆行信号，我们的初步结果已经确定了一个关键的转录共激活因子(TORC3)，它位于线粒体基质中，似乎在线粒体损伤后移位到细胞核。因此，目前应用的目的是：1)确定TORC3在线粒体中的定位，并与TORC2的定位进行比较。Torc 2和Torc 3是肝脏中的两个转录共激活因子，上调Hsp60和PGC-1a的表达。TORC2(细胞液)和TORC3(线粒体基质)分开的假设将被仔细测试，并使用差异或密度梯度离心法、有丝分裂和高分辨率共聚焦成像进行定义：2)在使用鱼藤酮、抗霉素A、目的：(A)研究线粒体氧化应激反应中TORC3的释放和核转位与生物发生基因表达的关系；(B)比较TORC2和TORC3对Hsp60启动子的核转位以及沉默TORC2和/或TORC3对MSR和线粒体生物发生的影响以及对致死性线粒体损伤的易感性；(C)确定CHOP及伴随的内质网应激与TORC2和TORC3相关的内质网应激对线粒体应激和生物发生反应的作用；这些研究将利用实时荧光聚合酶链式反应、蛋白质印迹法、芯片检测和siRNA技术；3)确定吡唑诱导的CYP2E1在MSR和线粒体体内生物发生中的作用；初步结果证实吡唑可诱导线粒体和ER CYP2E1并由此引起MSR；因此，吡唑治疗可为选择性胃内注射酒精的小鼠提供一种方便的体内线粒体损伤模型；4)描述MSR和线粒体生物发生基因的表达模式。这一应用的结果将令人信服地描述一种新的涉及TORC3转录共激活因子的从线粒体到细胞核的直接逆行信号通路，确定其相对于其他信号通路的作用，并开始建立其与体内酒精损伤的相关性。