The role of a novel AIF-associated nuclease PAAN1 in neuronal injury

新型 AIF 相关核酸酶 PAAN1 在神经元损伤中的作用

基本信息

批准号：
8545914
负责人：
Yingfei Wang
金额：
$ 9.48万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-15 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8545914
关键词：
Adult Amino Acids Animal Model Binding Bioinformatics Biological Assay Catalytic Domain Cause of Death Cell Death Cell Nucleus Cell Survival Cerebral Infarction Cessation of life Corpus striatum structure DNA DNA Damage DNA Fragmentation DNA Repair DNA biosynthesis Data Dependovirus Disease Educational process of instructing Enzymes Epigenetic Process Exonuclease Genomics Glutamates Goals Grant Hela Cells Histone H3 Histones Human Human body In Vitro Institutes Ischemic Neuronal Injury Knock-out Knockout Mice Label Laboratories Maps Mediating Mentors Modeling Molecular Morbidity - disease rate Names Neurologic Neurologic Dysfunctions Neuronal Injury Neurons Nuclear Nuclear Translocation Patients Phase Play Poly(ADP-ribose) Polymerases Post-Translational Protein Processing Protein Microchips Protein-Arginine N-Methyltransferase Proteins Quality of life Recruitment Activity Research Research Personnel Resistance Resources Role Serotyping Site Small Interfering RNA Stem cells Stroke Technology Testing Therapeutic Training Universities Wild Type Mouse Writing apoptosis inducing factor base cell type cellular engineering disability endonuclease high throughput screening histone methyltransferase histone modification human AMID protein in vivo innovation insight loss of function medical schools mortality mutant neuroprotection neurotoxicity novel novel therapeutics nuclease programs screening skills stem

项目摘要

DESCRIPTION (provided by applicant): Adult neurons are crucial and precious cell types controlling human body functions. Neuron injury following stroke and other neurologic diseases causes a significant loss of function. My long-term goal is to investigate the molecular mechanisms of neuronal injury and develop novel therapeutic strategies to treat patients suffering neurologic dysfunction. Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in glutamate neurotoxicity and cerebral infarction, which is a primary cause of subsequent morbidity and mortality following stroke. PARP-1 knockout mice are robustly resistant to stroke. Thus, it is critically important to understand the molecular mechanisms underlying PARP-1-dependent cell death (parthanatos) in stroke. Mitochondrial apoptosis-inducing factor (AIF) release and translocation to the nucleus causes chromatinolysis, which is the commitment point for parthanatos. However, little is known how AIF induces chromatinolysis and neuronal death after its nuclear translocation, since AIF itself has no endonuclease activity. Through an unbiased 17K human protein chip screening and a second siRNA-based PARP-1-dependent cell viability high-throughput screening, thirteen hits including AIF interactor 18 and protein arginine methyltransferase 6 (PRMT6) were identified. Here we focus on AIF interactor 18 as our preliminary data showed that knockdown of AIF interactor 18 is as protective as PARP-1 knockdown in parthanatos and it possesses hitherto unidentified endonuclease activity. Therefore, we name AIF interactor 18 as PAAN1 (Parthanatos-dependent AIF-Associated Nuclease 1). In the mentored K99 phase, we will first define the role of PAAN1 in ischemic neuronal death both in vitro and in vivo. We will then determine the specific PAAN1 endonuclease activity and determine if its endonuclease activity is required for neuronal injury. Histone post-translational modifications may regulate PAAN1 recognition in tightly wrapped genomic DNA to induce chromatinolysis and cell death after PARP-1 activation. PRMT6, a histone methyltransferase, was identified to be involved in parthanatos. In the independent R00 phase, I will study the role of PRMT6 in PAAN1-mediated ischemic cell death to understand the mechanism of PAAN1 recruitment to the DNA damage sites. I will further identify and characterize the functional PAAN1-interacting proteins required for parthanatos using two high-throughput screens, in order to understand how PAAN1 endonuclease activity is regulated by its networks. The successful completion of this project will yield important insights into cellular control of PAAN1 endonuclease activity and parthanatos and may provide new targets for developing innovative therapeutic approaches to treat patients with stroke. The mentored approach will be conducted in the laboratories of Drs. Valina and Ted Dawson and Dr. Raymond Koehler at the Johns Hopkins University School of Medicine. I have access to the resources available to Drs. Dawson and Koehler laboratories and the Neuroregeneration and Stem Cell Programs in Institute for Cell Engineering. To successfully accomplish the proposed research, during the mentored phase, I intend to take epigenetics courses and acquire skills in experimental stroke models and bioinformatics technologies, as well as grant writing and teaching skills. This training will allow me to expand my expertise and help my transition into a successful independent academic researcher.

描述（申请人提供）：成体神经元是控制人体功能的重要且珍贵的细胞类型。中风和其他神经系统疾病后的神经元损伤会导致功能显着丧失。我的长期目标是研究神经元损伤的分子机制，并开发新的治疗策略来治疗神经功能障碍患者。聚（ADP-核糖）聚合酶-1 (PARP-1) 在谷氨酸神经毒性和脑梗塞中发挥着关键作用，而脑梗塞是中风后发病和死亡的主要原因。 PARP-1 基因敲除小鼠对中风有很强的抵抗力。因此，了解中风中 PARP-1 依赖性细胞死亡 (parthanatos) 的分子机制至关重要。线粒体凋亡诱导因子（AIF）释放并易位至细胞核导致染色质溶解，这是parthanatos的承诺点。然而，由于 AIF 本身没有核酸内切酶活性，人们对 AIF 在核易位后如何诱导染色质分解和神经元死亡知之甚少。通过无偏倚的 17K 人类蛋白质芯片筛选和第二次基于 siRNA 的 PARP-1 依赖性细胞活力高通量筛选，鉴定出包括 AIF 相互作用子 18 和蛋白质精氨酸甲基转移酶 6 (PRMT6) 在内的 13 个命中目标。在这里，我们重点关注 AIF 相互作用蛋白 18，因为我们的初步数据表明，AIF 相互作用蛋白 18 的敲低与 PARP-1 敲低在 parthanatos 中具有相同的保护作用，并且它具有迄今为止未鉴定的核酸内切酶活性。因此，我们将 AIF 相互作用蛋白 18 命名为 PAAN1（Parthanatos 依赖性 AIF 相关核酸酶 1）。在 K99 指导阶段，我们将首先定义 PAAN1 在体外和体内缺血性神经元死亡中的作用。然后我们将确定特定的 PAAN1 核酸内切酶活性，并确定其核酸内切酶活性是否是神经元损伤所必需的。组蛋白翻译后修饰可能会调节紧密包裹的基因组 DNA 中的 PAAN1 识别，从而在 PARP-1 激活后诱导染色质分解和细胞死亡。 PRMT6 是一种组蛋白甲基转移酶，已被鉴定与 parthanatos 相关。在独立R00阶段，我将研究PRMT6在PAAN1介导的缺血性细胞死亡中的作用，以了解PAAN1招募到DNA损伤位点的机制。我将使用两个高通量筛选进一步鉴定和表征 parthanatos 所需的功能性 PAAN1 相互作用蛋白，以便了解 PAAN1 核酸内切酶活性如何受其网络调节。该项目的成功完成将为 PAAN1 核酸内切酶活性和 parthanatos 的细胞控制提供重要的见解，并可能为开发治疗中风患者的创新治疗方法提供新的目标。指导方法将在博士的实验室中进行。约翰·霍普金斯大学医学院的 Valina 和 Ted Dawson 以及 Raymond Koehler 博士。我可以访问博士可用的资源。道森和科勒实验室以及细胞工程研究所的神经再生和干细胞项目。为了成功完成拟议的研究，在指导阶段，我打算参加表观遗传学课程并获得实验性中风模型和生物信息学技术的技能，以及拨款写作和教学技能。这次培训将使我能够扩展我的专业知识，并帮助我转变为一名成功的独立学术研究员。