Cellular Mechanisms of Hepatotoxicity.

肝毒性的细胞机制。

• 批准号: 8818039
• 负责人: NEIL KAPLOWITZ
• 金额: $ 47.61万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 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/8818039
• 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; 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; liver injury; mitochondrial dysfunction; mutant; new therapeutic target; novel; prevent; public health relevance; src-Family Kinases; therapeutic target

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）：我们已经鉴定了c-jun-N-末端激酶（JNK）和线粒体外膜蛋白Sab之间的一种新型且极其重要的相互作用，其导致线粒体功能障碍和ROS产生增加，这反过来又在自放大循环中维持JNK活化，导致对乙酰氨基酚（APAP）引起的肝细胞坏死和TNF、ER应激或饱和脂肪酸引起的细胞凋亡。基于我们完成的工作和初步结果，我们准备推进我们对Sab在肝毒性中作用的理解。具体目标是：1）使用Sab敲除小鼠和突变的Sab形式的表达确定Sab的磷酸化在介导JNK依赖性肝毒性中的作用。假设：JNK介导的Sab磷酸化是线粒体功能受损和毒性所必需的。我们将在体外和体内验证肝特异性条件性敲除Sab在各种JNK依赖性毒性中具有保肝作用。然后，我们将表达突变的Sabs（磷酸化无能和磷酸化模拟突变体），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的去磷酸化，其使维持电子传递所需的持续需要的活性c-Src失活;这导致氧化磷酸化的抑制，并增加线粒体内活性c-Src的表达。 活性氧，JNK依赖性肝毒性的关键步骤。我们计划识别和定位线粒体中Src家族的调节子及其与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，我们将扩展研究线粒体Src失调从APAP到其他模型的JNK依赖性肝毒性在体外和体内。3)确定线粒体分裂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MFF的Sab依赖性稳定，允许DRP-1易位和线粒体分裂。我们有两个重要的初步发现。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以确认与Sab的关联并确定P-JNK是否磷酸化MFF。我们将确定是否蛋白酶体或线粒体内降解占低基础MFF。然后，我们将敲除MFF，并确定这是否提供针对JNK介导的线粒体功能障碍和损伤的保护。类似地，我们将评估敲低裂变中的另一个关键蛋白DRP-1的影响，并通过显性负性DRP-1的表达来证实这些发现。