Cellular Mechanisms of Hepatotoxicity.

描述（由申请人提供）：我们已经确定了c-jun- n-末端激酶（JNK）与线粒体、外膜蛋白Sab之间的一种新颖且极其重要的相互作用，这种相互作用导致线粒体功能障碍和ROS产生增加，进而维持JNK在自我放大周期中的激活，导致对乙酰氨基酚（APAP）导致肝细胞坏死，TNF、内质网应激或饱和脂肪酸导致肝细胞凋亡。基于我们完成的工作和初步结果，我们准备推进我们对Sab在肝毒性中的作用的理解。具体目的是：1)利用Sab敲除小鼠和Sab突变形式的表达，确定Sab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损伤和毒性所必需的。我们将在体外和体内验证肝脏特异性条件敲除Sab对各种JNK依赖性毒性具有肝保护作用。然后，我们将表达突变的Sabs（磷酸化不功能和拟磷突变体），并确定对线粒体功能和毒性易感性的影响。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：JNK对Sab的磷酸化导致线粒体内活性c-Src去磷酸化，使维持电子传递所需的活性c-Src失活；这导致氧化磷酸化的抑制和增加

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.