Cellular Mechanisms of Hepatotoxicity.

肝毒性的细胞机制。

基本信息

批准号：
9052172
负责人：
NEIL KAPLOWITZ
金额：
$ 47.65万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-10 至 2020-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9052172
关键词：
Accounting Acetaminophen Acute Liver Failure Address Agammaglobulinaemia tyrosine kinase Apoptosis Binding Binding Proteins Brefeldin A Cell Death Cell Line Cessation of life Complex Dactinomycin Data Docking Dominant-Negative Mutation Electron Transport Exposure to Family Functional disorder Funding Galactosamine Hepatocyte Hepatotoxicity Impairment In Vitro Injury Knock-out Knockout Mice Lead Liver Liver Mitochondria LoxP-flanked allele MAPK8 gene Mediating Membrane Proteins Mitochondria Modeling Mus Mutate N-terminal Outer Mitochondrial Membrane Oxidative Phosphorylation Palmitic Acids Pathway interactions Phosphorylation Phosphotransferases Predisposition Production Protein Dephosphorylation Proteins Research Respiration Role SRC gene Saturated Fatty Acids Serine Phosphorylation Site Signal Pathway Site-Directed Mutagenesis Stress Surface TNF gene Toxic effect Tunicamycin Work base hepatic necrosis in vivo knock-down liver injury mitochondrial dysfunction mutant new therapeutic target novel prevent public health relevance src-Family Kinases therapeutic target

项目摘要

DESCRIPTION (provided by applicant): We have identified a novel and extremely important interplay between c-jun-N-terminal kinase (JNK) and mitochondria, outer membrane protein Sab, which leads to mitochondria dysfunction and increased ROS production, which in turn sustains JNK activation in a self-amplifying cycle, leading to hepatocellular necrosis from acetaminophen (APAP) and apoptosis from TNF, ER stress, or saturated fatty acids. Based upon our work accomplished and preliminary results we are poised to advance our understanding of the role of Sab in hepatotoxicity. The specific aims are: 1) Determine the role of phosphorylation of Sab in mediating JNK dependent hepatotoxicity using Sab knockout mice and expression of mutated Sab forms. Hypothesis: JNK mediated phosphorylation of Sab is required for impairment of mitochondrial function and toxicity. We will verify that liver specific conditional knockout of Sab is hepatoprotective in various JNK dependent toxicities in vitro and in vivo. We will then express mutated Sabs (phosphorylation incompetent and phosphomimetic mutants) and determine the impact on mitochondrial function and susceptibility to toxicity. 2) Determine the intramitochondrial signaling pathway downstream of Sab: role of mitochondrial c-Src and Dok-4. Hypothesis: JNK phosphorylation of Sab leads to dephosphorylation of intramitochondrial active c-Src which inactivates the continuously required active c-Src needed to maintain electron transport; this leads to inhibition of oxidative phosphorylation and increased ROS, key steps in JNK-dependent hepatotoxicities. We plan to identify and localize the regulators of the Src family in mitochondria and their association with Sab and Dok-4 (mitochondrial Src kinase docking protein) and the role of Src dysregulation in mitochondrial ROS production. Then we will extend out studies of mitochondrial Src dysregulation from APAP to other models of JNK dependent hepatotoxicity in vitro and in vivo. 3) Determine the mechanism and importance of mitochondrial fission and its interplay with Sab and JNK in mediating hepatotoxicity. Hypothesis: JNK mediated hepatotoxicity depends on the Sab dependent stabilization of MFF, allowing DRP-1 translocation and mitochondrial fission. We have made two important preliminary discoveries. The outer membrane Mitochondrial Fission Factor, MFF, co-immunoprecipitates (IP) with Sab and its expression rapidly increases after APAP. Therefore, we will IP MFF to confirm the association with Sab and determine if P-JNK phosphorylates MFF. We will determine if proteasomal or intramitochondrial degradation accounts for low basal MFF. Then we will knockdown MFF and determine if this affords protection against JNK mediated mitochondrial dysfunction and injury. Similarly, we will assess the effect of knockdown of the other key protein in fission, DRP-1, and confirm the findings by expression of dominant negative DRP-1.

描述（由申请人提供）：我们已经确定了 c-jun-N 末端激酶 (JNK) 和线粒体、外膜蛋白 Sab 之间的一种新颖且极其重要的相互作用，它会导致线粒体功能障碍和 ROS 产生增加，进而维持线粒体功能。 JNK 在自我放大循环中激活，导致对乙酰氨基酚 (APAP) 引起的肝细胞坏死以及 TNF、ER 应激或饱和脂肪酸引起的细胞凋亡。根据我们已完成的工作和初步结果，我们准备进一步了解 Sab 在肝毒性中的作用。具体目标是： 1) 使用 Sab 敲除小鼠和突变 Sab 形式的表达确定 Sab 磷酸化在介导 JNK 依赖性肝毒性中的作用。假设：JNK 介导的 Sab 磷酸化是损害线粒体功能和毒性所必需的。我们将在体外和体内验证肝脏特异性条件性敲除 Sab 在各种 JNK 依赖性毒性中具有肝保护作用。然后，我们将表达突变的 Sabs（磷酸化无能和磷酸化突变体）并确定对线粒体功能和毒性敏感性的影响。 2)确定Sab下游的线粒体内信号通路：线粒体c-Src和Dok-4的作用。假设：Sab 的 JNK 磷酸化导致线粒体内活性 c-Src 去磷酸化，从而使维持电子传输所需的持续所需的活性 c-Src 失活；这导致氧化磷酸化的抑制并增加 ROS，JNK 依赖性肝毒性的关键步骤。我们计划鉴定并定位线粒体中 Src 家族的调节因子及其与 Sab 和 Dok-4（线粒体 Src 激酶对接蛋白）的关联，以及 Src 失调在线粒体 ROS 产生中的作用。然后我们将把线粒体 Src 失调的研究从 APAP 扩展到 JNK 依赖性肝毒性的体外和体内其他模型。 3) 确定线粒体裂变的机制和重要性及其与 Sab 和 JNK 在介导肝毒性中的相互作用。假设：JNK 介导的肝毒性依赖于 Sab 依赖性的 MFF 稳定，从而允许 DRP-1 易位和线粒体裂变。我们取得了两项重要的初步发现。外膜线粒体裂变因子 (MFF) 与 Sab 共免疫沉淀 (IP)，其表达在 APAP 后迅速增加。因此，我们将IP MFF 来确认与Sab 的关联并确定P-JNK 是否磷酸化MFF。我们将确定蛋白酶体或线粒体内降解是否导致基础 MFF 较低。然后我们将敲低 MFF 并确定这是否可以提供针对 JNK 介导的线粒体功能障碍和损伤的保护。同样，我们将评估敲低裂变中另一个关键蛋白 DRP-1 的效果，并通过显性失活 DRP-1 的表达来证实这一发现。