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（由申请人提供）：成人神经元是控制人体功能的至关重要和宝贵的细胞类型。中风后的神经元损伤和其他神经系统疾病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，并制定新的治疗策略来治疗患有神经功能障碍的患者。聚（ADP-核糖）聚合酶1（PARP-1）在谷氨酸神经毒性和脑梗塞中起关键作用，这是中风后随后发病率和死亡率的主要原因。 PARP-1敲除小鼠对中风具有耐药性。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。线粒体凋亡因子（AIF）释放并转移到细胞核会导致染色质溶解，这是parthanatos的承诺点。然而，由于AIF本身没有核酸内切酶活性，因此几乎鲜为人知的AIF如何在其核转运后诱导染色质溶解和神经元死亡。通过公正的17K人蛋白芯片筛选和第二个基于siRNA的PARP-1依赖性细胞活力高通量筛选，确定了13个命中，包括AIF相互作用18和蛋白精氨酸甲基转移酶6（PRMT6）。在这里，我们专注于AIF Interactor 18，因为我们的初步数据表明，AIF Interactor 18的敲低与Parthanatos中的PARP-1敲低一样保护性，并且它具有迄今为止未知的核酸内切酶活性。因此，我们将AIF相互作用的18个称为PAAN1（依赖于AIF的AIF相关核酸酶1）。在指导的K99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，并确定其神经元损伤是否需要其内切清除酶活性。组蛋白的翻译后修饰可能调节紧密包裹的基因组DNA中的PAAN1识别，以诱导PARP-1激活后的染色质溶解和细胞死亡。 PRMT6是一种组蛋白甲基转移酶，被认为与parthanatos有关。在独立的R00阶段，我将研究PRMT6在PAAN1介导的缺血细胞死亡中的作用，以了解PAAN1募集到DNA损伤位点的机制。我将进一步识别并表征使用两个高通量屏幕所需的parthanatos所需的功能性PAAN1相互作用蛋白，以了解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产生对PAAN1核酸内切酶活性和parthanatos的细胞控制的重要见解，并可能为开发创新的治疗方法提供治疗中风患者的新目标。指导的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瓦利娜（Valina）和泰德·道森（Ted Dawson）和雷蒙德·科勒（Raymond Koehler）博士。我可以访问DRS可用的资源。 Dawson和Koehler Laboratories以及细胞工程研究所的神经变性和干细胞程序。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，并在实验中风模型和生物信息学技术中获得技能，以及赠款写作和教学技能。这项培训将使我能够扩大自己的专业知识，并帮助我的过渡到成功的独立学术研究员。