Targets of JNK in acute hepatotoxicity.

JNK 急性肝毒性的靶点。

• 批准号: 10098174
• 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/10098174
• 关键词: Acetaminophen; Acute; 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; 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; Pharmaceutical Preparations; 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; base; body system; 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.

摘要 持续活化的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。该建议集中在两个新的JNK靶向蛋白，细胞质 γ-谷氨酰半胱氨酸连接酶（GCL）和线粒体外膜SAB（SH 3BP 5）介导的线粒体膜电位改变 通过促进MAPK激酶的活性氧（ROS）物质依赖性活化来持续JNK活化 级联。基于初步的数据，我们提出：（a）P-JNK靶向GCLC亚基进行快速蛋白水解， 增强ROS暴露和随后的MAPK级联激活的降解;（B）特异性P-JNK SAB上的对接和磷酸化位点介导SAB依赖的线粒体ROS产生， （c）具有GCL降解的SAB 2的第二同种型维持P-JNK-mitoSAB-ROS活化环; 修饰的N-末端不能在线粒体内转导效应，但保留C-末端的能力 是P-JNK底物，因此是潜在的诱饵。我们的总体假设是，P-JNK靶向 两个新靶点GCL和SAB的磷酸化介导持续的JNK激活， 对乙酰氨基酚（APAP）在其他模型中通过两种机制引起的肝毒性和细胞凋亡 SAB依赖性持续P-JNK，然后调节凋亡调节因子。因此，提案的目的是 分别是：（1）确定JNK在γ-谷氨酰半胱氨酸连接酶（GCL）亚基调节中的作用及其影响 对乙酰氨基酚（APAP）肝毒性中GSH恢复的影响：初步结果表明，GCLC APAP处理后，JNK活性降低，而JNK抗性表达 在APAP诱导的肝损伤中，突变的GCLC在坏死发生之前抑制P-JNK。GCLC和GCLM 含有P-JNK对接位点（KIM，激酶相互作用基序）和可能的磷酸化位点，但只有GCLC 含有PEST切割位点。(2)表征特异性JNK结合和磷酸化位点的作用， SAB介导JNK依赖性毒性及其与SAB寡聚化的关系：SAB含有两种可能的P-JNK 对接（KIM）位点和四个丝氨酸（SP/L）位点用于磷酸化。我们将进行定点突变 阐明SAB依赖性信号传导至线粒体内的关键环节。我们 评估SAB的磷酸化对SAB的同源或异源寡聚化的依赖性及其在 促进线粒体内信号转导。(3)定义两种异构体的表达和功能， 小鼠和人肝脏中的SAB（SAB 1和-2）：我们鉴定了SAB的两种同种型。小鼠SAB 2具有独特的 N-末端氨基酸序列，而人SAB 2与人SAB 2相比更短并且在N-末端截短。 SAB1.初步数据表明，小鼠SAB 2位于线粒体内，不发出信号， 线粒体，并且，当在野生型小鼠中过表达时，保护免受肝损伤，这表明它作为一种抗氧化剂。 诱饵基质