Targets of JNK in acute hepatotoxicity.

JNK 急性肝毒性的靶点。

基本信息

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
项目类别：
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

项目摘要

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发挥着关键作用该提案重点关注两种新型 JNK 靶向蛋白：细胞质。 -谷氨酰半胱氨酸连接酶（GCL）和线粒体外膜SAB（SH3BP5）及其介导作用通过促进活性氧 (ROS) 物种依赖性 MAPK 激酶激活来持续激活 JNK 根据初步数据，我们建议 (a) P-JNK 靶向 GCLC 亚基进行快速蛋白水解。降解，增强 ROS 暴露和随后的 MAPK 级联激活 (b) 特异性 P-JNK； SAB 上的对接和磷酸化位点介导 SAB 依赖性线粒体 ROS 产生并共同介导随着 GCL 降解，维持 P-JNK-mitoSAB-ROS 激活环；(c) SAB2 的第二种亚型，具有修饰的 N 末端无法在线粒体内转导效应，但保留了 C 末端的能力是 P-JNK 底物，因此是一个潜在的诱饵。我们的首要假设是 P-JNK 靶向。两个新靶标 GCL 和 SAB 的磷酸化介导持续的 JNK 激活，从而导致对乙酰氨基酚（APAP）的肝毒性有两个作用机制，以及其他模型中的细胞凋亡 SAB 依赖性持续 P-JNK，然后调节细胞凋亡调节因子，因此，该提案的目的是。分别是：（1）确定JNK对-谷氨酰半胱氨酸连接酶（GCL）亚基的调节作用及影响对乙酰氨基酚 (APAP) 肝毒性中 GSH 回收率的影响：初步结果表明 GCLC 可以快速 APAP 治疗后降解，同时 JNK 持续激活，同时 JNK 表达耐药在 APAP 诱导的肝损伤中，突变的 GCLC 会在坏死发生前抑制 P-JNK。包含 P-JNK 对接位点（KIM，激酶相互作用基序）和可能的磷酸化位点，但仅包含 GCLC (2) 表征特定 JNK 结合和磷酸化位点的作用 SAB 介导 JNK 依赖性毒性以及与 SAB 寡聚化的关系：SAB 包含两种可能的 P-JNK 我们将进行定点诱变。阐明在线粒体内部 SAB 依赖性信号转导中哪些是不可缺少的。评估 SAB 磷酸化对 SAB 同源或异源寡聚化的依赖性及其在 (3) 定义两个异构体的表达和功能小鼠和人类 SAB（SAB1 和 -2）在肝脏中：我们鉴定出两种 SAB 亚型，小鼠 SAB2 具有独特的特性。 N 端氨基酸序列，而人 SAB2 与 SAB1。初步数据表明小鼠SAB2位于线粒体中，在内部不转导信号。线粒体，并且当在野生型小鼠中过度表达时，可以防止肝损伤，这表明它可以作为诱饵底物。