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。

基本信息

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
项目类别：
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

项目摘要

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。线粒体稳态的一个未经探索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）和线粒体生物发生，以响应线粒体应激（例如，蛋白质折叠或氧化修饰），就像酒精引起的那样。 MSR 和生物发生反应主要由核基因的上调介导。我们已经开始探索这些反应的上游传感器和触发因素，称为线粒体逆行信号传导，我们的初步结果已经确定了一个关键的转录辅激活因子（TORC3），它驻留在线粒体基质中，并且似乎在线粒体逆行信号传导后易位到细胞核。损害。因此，本申请的目的是：1）确认TORC3在线粒体中的定位并与TORC2的定位进行比较。 TORC 2 和 3 是肝脏中的两个转录共激活因子，可上调 Hsp60 和 PGC-1a。将使用差速或密度梯度离心、线粒体和高分辨率共聚焦成像来仔细测试 TORC2（细胞质）和 TORC3（线粒体基质）分开的假设：2）检查 TORC3 在 MSR 和线粒体生物合成中的作用使用鱼藤酮、抗霉素 A 或线粒体中 Cyp2e1 表达的线粒体损伤细胞培养模型（HepG2 细胞和原代小鼠肝细胞） (a) 表征 TORC3 从线粒体释放的详细时间过程以及与响应线粒体氧化应激的 MSR 和生物发生基因表达相关的核转位 (b) 比较 TORC2 与 TORC3 到 Hsp60 启动子的核转位以及沉默 TORC2 和/或 TORC3 对 MSR 和线粒体生物发生以及对致命线粒体损伤的易感性； (c)确定与TORC2和TORC3相关的CHOP和伴随的ER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 PCR、蛋白质印迹、ChIP 测定和 siRNA； 3）明确吡唑诱导的Cyp2e1对体内MSR和线粒体生物合成的影响；初步结果证实吡唑诱导线粒体和 ER Cyp2e1 并导致 MSR；因此，吡唑治疗可能为选择性线粒体损伤提供方便的体内模型； 4)描述慢性胃内酒精喂养小鼠中MSR和线粒体生物发生基因的表达模式。该应用的结果应该令人信服地描述了涉及TORC3转录共激活子的从线粒体到细胞核的新型直接逆行信号通路的特征，定义了其相对于其他信号通路的作用，并开始确定其与体内酒精损伤的相关性。