Targets of JNK in acute hepatotoxicity.

JNK 急性肝毒性的靶点。

• 批准号: 10630057
• 负责人: NEIL KAPLOWITZ
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630057
• 关键词: Acute Liver Failure; Address; Amino Acid Sequence; Apoptosis; Apoptosis Regulation Gene; Apoptotic; Autoimmune Hepatitis; Binding Sites; Calpain; Catalytic Domain; Cell Death; Cessation of life; Cysteine; Cytoplasm; Data; Dependence; Disease; Docking; Enzymes; Family; GCLC gene; GCLM gene; Gene Expression; Glutathione; Health; Hepatocyte; Hepatotoxicity; Homo; Human; Impairment; Individual; Investigation; Knockout Mice; Ligase; Liver; MAP Kinase Gene; MAPK8 gene; Mediating; Membrane Proteins; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Modeling; Morbidity - disease rate; Mouse Strains; Mus; Mutate; N-terminal; Necrosis; Organ; Outer Mitochondrial Membrane; Oxidative Stress; Oxygen; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Predisposition; Production; Protein Isoforms; Proteins; Publishing; Reactive Oxygen Species; Recovery; Regulation; Resistance; Role; Seminal; Serine; Signal Transduction; Site; Site-Directed Mutagenesis; Stress; Testing; Toxic effect; Viral hepatitis; Wild Type Mouse; Work; acetaminophen-induced liver injury; acute hepatotoxicity; body system; drug induced liver injury; humanized mouse; knock-down; liver injury; mitochondrial dysfunction; mortality; multicatalytic endopeptidase complex; novel; novel strategies; overexpression; prevent; response; restoration; transcription factor

ABSTRACT Sustained activated c-Jun-N-terminal kinase (JNK) plays a pivotal role in mediating hepatotoxic cell death due to its phosphorylation of target proteins. This proposal focuses on two novel JNK targeted proteins, cytoplasmic - glutamyl cysteine ligase (GCL) and mitochondrial outer membrane SAB (SH3BP5) and their roles in mediating sustained JNK activation by promoting reactive oxygen (ROS) species dependent activation of the MAPK kinase cascade. Based on preliminary data we propose that (a) P-JNK targets GCLC subunit for rapid proteolytic degradation which enhances ROS exposure and consequent activation of MAPK cascade; (b) specific P-JNK docking and phosphorylation sites on SAB mediate SAB-dependent mitochondrial ROS production and together with GCL degradation sustain a P-JNK-mitoSAB-ROS activation loop; (c) a second isoform of SAB2 with a modified N-terminus is incapable of transducing an effect inside the mitochondria but retains ability of C-terminus to be a P-JNK substrate and therefore is a potential decoy. Our overarching hypothesis is that P-JNK targeting and phosphorylation of both novel targets, GCL and SAB, mediates sustained JNK activation leading to hepatotoxicity of acetaminophen (APAP) in a two-pronged mechanism and apoptosis in other models through SAB-dependent sustained P-JNK which then modulates apoptosis regulators. Thus, the aims of the proposal are: (1) Determine the role of JNK in the regulation of - glutamyl cysteine ligase (GCL) subunits and the impact on GSH recovery in acetaminophen (APAP) hepatotoxicity: Preliminary results show that GCLC is rapidly degraded after APAP treatment in conjunction with sustained JNK activation while expression of JNK resistant mutated GCLC dampens P-JNK before onset of necrosis in APAP induced liver injury. Both GCLC and GCLM contain a P-JNK docking site (KIM, kinase interaction motif) and possible phosphorylation sites but only GCLC contains a PEST cleavage site. (2) Characterize the role of specific JNK binding and phosphorylation sites of SAB in mediating JNK-dependent toxicity and relation to SAB oligomerization: SAB contains two possible P-JNK docking (KIM) sites and four serine (SP/L) sites for phosphorylation. We will perform site directed mutagenesis to elucidate which is/are indispensable in SAB-dependent signal transduction to inside of mitochondria. We assess the dependence of phosphorylation of SAB on the homo or hetero oligomerization of SAB and its role in promoting intramitochondrial signal transduction. (3) Define the expression and function of the two isoforms of mouse and human SAB (SAB1 and -2) in liver: We identified two isoforms of SAB. Mouse SAB2 has a unique N-terminal amino acid sequence whereas human SAB2 is shorter and truncated at N-terminus compared to SAB1. Preliminary data indicates that mouse SAB2 is in mitochondria, does not transduce a signal inside mitochondria, and, when overexpressed in wild type mice, protects against liver injury, suggesting it acts as a decoy substrate.

抽象的 持续激活的C-Jun-N-末端激酶（JNK）在介导肝毒性细胞死亡中起关键作用 靶蛋白的磷酸化。该提案重点介绍了两个新型的JNK靶向蛋白质细胞质 -谷氨酸半胱氨酸连接酶（GCL）和线粒体外膜SAB（SH3BP5）及其在中介中的作用 通过促进MAPK激酶的活性氧（ROS）依赖性激活来持续JNK激活 级联。根据初步数据，我们建议（a）P-JNK靶向GCLC亚基快速蛋白水解 降解可增强ROS暴露并随之而来的MAPK级联激活； （b）特定的p-jnk SAB上的对接和磷酸化位点介导SAB依赖性线粒体ROS的产生，一起 随着GCL降解，p-jnk-mitosab-ros激活环均维持； （c）SAB2的第二个同工型 修改的N末端无法传递线粒体内部的效果，但保留了C末端的能力 成为P-JNK底物，因此是潜在的诱饵。我们的总体假设是p-jnk定位 GCL和SAB的两个新靶标的磷酸化介导了JNK激活，导致JNK激活导致 在其他模型中，对乙酰氨基酚（APAP）的肝毒性（APAP）通过 依赖SAB的持续P-JNK，然后调节细胞凋亡调节剂。那，提案的目的 为：（1）确定JNK在调节gl-谷氨酸半胱氨酸连接酶（GCL）亚基中的作用和影响 关于对乙酰氨基酚（APAP）肝毒性的GSH恢复：初步结果表明GCLC迅速 APAP处理后与持续的JNK激活结合进行降解，而JNK抗性的表达 突变的GCLC在APAP诱导肝损伤的坏死发作之前会抑制P-JNK。 GCLC和GCLM都 包含一个P-JNK对接位点（KIM，激酶相互作用基序）和可能的磷酸化位点，但仅GCLC 包含一个有害生物的裂解部位。 （2）表征特定JNK结合和磷酸化位点的作用 SAB介导JNK依赖性毒性和与SAB低聚的关系：SAB包含两个可能的P-JNK 对接（KIM）位点和四个串行（SP/L）位点用于磷酸化。我们将执行定向诱变 阐明在SAB依赖性的信号转移到线粒体内部的必不可少的。我们 评估SAB的光磷酸化对SAB的同型或杂音寡聚及其在其在其中的作用的依赖性 促进Intochrial信号转导。 （3）定义两个同工型的表达和功能 肝脏中的小鼠和人类SAB（SAB1和-2）：我们确定了SAB的两种同工型。鼠标SAB2具有独特的 N末端氨基酸序列，而人类SAB2在N末端较短和截断 SAB1。初步数据表明鼠标SAB2在线粒体中，不会在内部翻译信号 线粒体，当在野生型小鼠中过表达时，可以防止肝损伤，表明它充当 诱饵底物。