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（RIPK1和RIPK3）是参与细胞死亡和存活的蛋白质。RIPK1在调节这些结果中的作用是由环境和蛋白的激酶与支架功能决定的。坏死坏死是一种依赖于RIPK1激酶功能的受调控的细胞死亡形式，当半胱天酶被抑制时，它激活RIPK1- ripk3坏死体，导致MLKL的激活和细胞膜破裂。在对乙酰氨基酚（APAP）肝损伤模型中，由于RIPK3-/-和MLKL -/-小鼠没有受到保护，我们发现坏死坏死没有作用。然而，我们发现了RIPK1在APAP毒性中的一种新的脚手架功能。我们已经证明，敲除RIPK1可以保护小鼠免受APAP肝毒性上游JNK的影响，JNK是APAP诱导细胞死亡的关键介质。有趣的是，我们的初步研究表明RIPK1激酶死亡的转基因小鼠不受APAP的保护，这强烈表明该蛋白在细胞死亡信号传导中具有支架功能。此外，我们发现肝脏RIPK3蛋白具有细胞特异性分布，在包括肝窦内皮细胞（LSECs）在内的非实质细胞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的职业发展建议的基础：APAP肝毒性和缺血再灌注损伤（IRI）。RIPK1通过肝细胞中的支架功能和LSECs中的激酶功能调节肝脏细胞死亡。在该奖项的前两年，我们将重点关注aim1，该项目将系统地探索RIPK1在肝细胞中的支架功能。我们还将确定与RIPK1相互作用的蛋白，以及RIPK1在APAP毒性和其他内质网应激模型中导致JNK激活的信号通路中的作用。随后，在该提案的第二个目的中，我们将通过检查条件肝细胞或内皮细胞缺失小鼠的肝损伤，探索细胞类型特异性缺失RIPK1和RIPK3在肝损伤模型中的影响。拟议的研究是为Lily Dara博士颁发的指导临床科学家发展奖（K08）的核心组成部分，围绕该奖项，Dara博士建立了一个结构化的项目。Dara博士已经确定了在整个K奖期间进行额外训练的四个目标领域：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年的职业发展计划，并组建了一个多学科的专家咨询团队，专门研究这些核心组成部分，以指导她的工作并批评她的发现。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绝佳资源，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与合作的机会。该项目未来五年的直接重点是RIPK1和RIPK3在急性肝损伤模型中的作用。在完成这些研究后，Dr. Dara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。该提案中计划的实验，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Dara博士成功过渡到独立的医生科学家，同时推进对RIP激酶在肝脏疾病中的作用的理解。

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.