Targets of JNK in acute hepatotoxicity.

摘要 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 它对目标蛋白的磷酸化。这项建议关注两个新的JNK靶向蛋白，细胞质 -谷氨酰半胱氨酸连接酶和线粒体外膜SAB及其在信号转导中的作用 通过促进活性氧(ROS)对MAPK激酶的激活来维持JNK的激活 卡斯卡德。根据初步数据，我们提出：(A)P-JNK以GCLC亚基为靶点进行快速蛋白降解 增加ROS暴露和随后激活MAPK级联的降解；(B)特异性P-JNK SAB上的对接和磷酸化位点共同介导SAB依赖的线粒体ROS的产生 随着GCL的降解，维持一个P-JNK-mitoSAB-ROS激活环；(C)SAB2的第二个亚型，具有 修饰的N-末端不能在线粒体内传递效应，但保留了C-末端的能力 作为P-JNK底物，因此是一个潜在的诱饵。我们的主要假设是P-JNK靶向 而这两个新靶点GCL和SAB的磷酸化介导了JNK的持续激活，导致 对乙酰氨基酚(APAP)双管齐下的肝毒性和其他模型中的细胞凋亡 SAB依赖的持续P-JNK随后调节细胞凋亡调节。因此，该提案的目的是 主要有：(1)确定JNK在调节-谷氨酰半胱氨酸连接酶(GCL)亚基中的作用及其影响 对乙酰氨基酚(APAP)肝毒性中GSH的恢复：初步结果显示GCLC迅速 APAP处理联合JNK持续激活后降解，而JNK表达耐药 突变型GCLC在APAP肝损伤发病前抑制P-JNK。GCLC和GCLM 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，但仅包含GCLC 包含一个害虫裂解部位。(2)鉴定JNK特异性结合位点和磷酸化位点的作用 SAB在介导JNK依赖毒性中的作用及其与SAB寡聚的关系：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 阐明在SAB依赖的信号转导到线粒体内部的过程中哪些是不可缺少的。我们 评估SAB的磷酸化对SAB的同源或异源齐聚的依赖性及其在 促进线粒体内信号转导。(3)定义两种异构体的表达和功能 小鼠和人肝组织中的SAB(SAB1和-2)：我们鉴定了SAB的两种亚型。小鼠SAB2有一种独特的 而人SAB2的N-末端氨基酸序列更短，N-末端截短 SAB1。初步数据显示，小鼠SAB2位于线粒体内，不传递信号 线粒体，当在野生型小鼠中过度表达时，可以保护肝脏免受损伤，这表明它作为一种 诱饵衬底。

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.