Role of RIPK1 and RIPK3 in liver injury.

描述（由申请人提供）：受体相互作用蛋白激酶（RIPK 1和RIPK 3）是参与细胞死亡和存活的蛋白质。RIPK 1在调节这些结果中的作用由背景和蛋白质的激酶与支架功能决定。坏死性凋亡是一种依赖于RIPK 1激酶功能的调节性细胞死亡形式，激活RIPK 1-RIPK 3坏死体，导致MLKL激活和半胱天冬酶抑制时细胞膜破裂。使用对乙酰氨基酚（APAP）肝损伤模型，我们发现坏死性凋亡没有作用，因为RIPK 3-/-和MLKL -/-小鼠没有受到保护。然而，我们发现了一种新的支架功能RIPK 1在APAP毒性。我们已经表明，敲低RIPK 1可以保护小鼠免受JNK上游的APAP肝毒性，JNK是APAP诱导细胞死亡的关键介质。有趣的是，我们的初步研究显示RIPK 1激酶死亡的转基因小鼠并没有受到APAP的保护，这强烈表明该蛋白在细胞死亡信号传导中的支架功能。此外，我们还发现了肝RIPK 3蛋白的细胞特异性分布，在非实质细胞，包括肝窦内皮细胞（LSECs）中有丰富的蛋白表达。这些发现导致了以下总体假设，这构成了两种临床相关肝损伤模型中的职业发展建议的基础：APAP肝毒性和缺血再灌注损伤（IRI）。RIPK 1通过肝细胞中的支架功能和LSEC中的激酶功能调节肝脏中的细胞死亡。在该奖项的前两年，我们将专注于aim 1，它将系统地探索RIPK 1在肝细胞中的支架功能。我们还将确定与RIPK 1相互作用的蛋白质以及RIPK 1在APAP毒性和其他ER应激模型中导致JNK激活的信号通路中的作用。随后，在该提案的第二个目标中，我们将通过检查条件性肝细胞或内皮缺失小鼠的肝损伤来探索RIPK 1和RIPK 3的细胞类型特异性缺失在肝损伤模型中的作用。拟议的研究是Lily达拉博士指导临床科学家发展奖（K 08）的核心组成部分，围绕该奖项，为达拉博士建立了一个结构化计划。达拉博士已经确定了四个目标领域的额外培训在整个K奖期间：蛋白质生物化学和信号，转基因小鼠模型，LSEC病理生物学，和肝脏缺血再灌注损伤模型。为了实现这些目标，她设计了一个5年职业发展计划，并组建了一个多学科的科学家咨询团队，专门从事这些核心组成部分，以指导她的工作和批评她的发现。南加州大学在肝脏研究方面有着悠久的传统。NIDDK资助的南加州大学肝病研究中心是候选人的优秀资源，通过其核心提供实用的设施和设备，并通过其庞大的会员基础提供科学交流和合作的机会。该项目在未来五年的直接重点是RIPK 1和RIPK 3在急性肝损伤模型中的作用。完成这些研究后，达拉博士的长期目标是将这些发现扩展到慢性肝损伤的实验模型，如酒精性和非酒精性脂肪性肝病/脂肪性肝炎。本提案中计划的实验、导师咨询委员会的指导以及南加州大学理想的培训环境将确保达拉博士成功转变为独立的医生-科学家，同时促进对RIP激酶在肝病中作用的理解。

DESCRIPTION (provided by applicant): The Receptor Interacting Protein Kinases (RIPK1 and RIPK3) are proteins involved in cell death and survival. The role of RIPK1 in regulating these outcomes is determined by context and the kinase versus scaffolding function of the protein. Necroptosis is a form of regulated cell death dependent on the kinase function of RIPK1, activating the RIPK1-RIPK3 necrosome leading to the activation of MLKL and cell membrane rupture when caspases are inhibited. Using the acetaminophen (APAP) liver injury model, we found no role for necroptosis as RIPK3-/- and MLKL -/- mice are not protected. However, we identified a novel scaffolding function for RIPK1 in APAP toxicity. We have shown that knocking down RIPK1 protects mice from APAP hepatotoxicity upstream of JNK, a key mediator of APAP induced cell death. Interestingly, our preliminary studies reveal RIPK1 kinase-dead transgenic mice are not protected from APAP, strongly suggesting a scaffolding function of the protein in cell death signaling. In addition, we have found a cell specific distribution for hepatic RIPK3 protein, with abundant protein expression in the non-parenchymal cells, including liver sinusoidal endothelial cells (LSECs). These findings have lead to the following overarching hypothesis, which forms the basis of this career development proposal in two clinically relevant liver injury models: APAP hepatotoxicity, and ischemia reperfusion injury (IRI). RIPK1 regulates cell death in the liver through a scaffolding function in hepatocytes and a kinase function in LSECs. For the first two years of the award we will focus on aim1, which will systematically explore the scaffolding function of RIPK1 in hepatocytes. We will also determine the interacting proteins with RIPK1 and the role of RIPK1 in the signaling pathway that leads to JNK activation in APAP toxicity and other ER stress models. Subsequently, in the second aim of the proposal we will explore the effect of cell type specific deletion of RIPK1 and RIPK3 in models of liver injury by examining liver injury in mice with conditional hepatocyte or endothelial deletions. The proposed studies are the core components of the Mentored Clinical Scientist Development Award (K08) for Dr. Lily Dara, around which a structured program for Dr. Dara has been built. Dr. Dara has identified four target areas for additional training throughout the K Award period: protein biochemistry and signaling, transgenic mice models, LSEC pathobiology, and the liver ischemia reperfusion injury model. To achieve these goals, she has devised a 5-year career development plan and assembled a multidisciplinary advisory team of scientists specializing in each of these core components to guide her work and critique her findings. The University of Southern California has a long-standing tradition in excellence in liver research. The NIDDK- funded USC Research Center for Liver Disease is an excellent resource for the candidate, providing both practical facilities and equipment through its cores, and the opportunity for scientific exchange and collaboration through its large membership base. The immediate focus of this project in the next five years is the role of RIPK1 and RIPK3 in models of acute liver injury. After completion of these studies the long-term goal of Dr. Dara is to extend these findings to experimental models of chronic liver injury such as alcoholic and nonalcoholic fatty liver disease/steatohepatitis. The experiments planned in this proposal, the guidance of the mentorship advisory committee, and the ideal training environment at USC will ensure Dr. Dara's successful transition to an independent physician-scientist while advancing the understanding of the role of RIP kinases in liver disease.