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

• 批准号: 8441224
• 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
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8441224
• 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; Screening procedure; 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; skills; stem

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. PUBLIC HEALTH RELEVANCE: Stroke is the second cause of death and disability in the world. Parthanatos following stroke or other neurologic diseases is a primary cause for the subsequent death, disability and loss of quality of life. Understanding the mechanisms underlying parthanatos will offer innovative therapeutic approaches to treat patients with stroke or other neurologic diseases.

描述(申请人提供)：成年神经元是控制人体功能的关键和宝贵的细胞类型。中风和其他神经系统疾病后的神经元损伤会导致严重的功能丧失。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。多聚(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，而脑梗塞是卒中后继发发病率和死亡率的主要原因。PARP-1基因敲除小鼠对中风有很强的抵抗力。因此，了解卒中中依赖PARP-1的细胞死亡(副死亡症)的分子机制至关重要。线粒体凋亡诱导因子(AIF)的释放和移位导致核染色质溶解，这是肝细胞死亡的承诺点。然而，由于AIF本身没有核酸内切酶活性，因此AIF在其核转位后如何诱导染色质溶解和神经元死亡的机制还知之甚少。通过无偏的17K人蛋白芯片筛选和第二次基于siRNA的依赖PARP-1的细胞活性高通量筛选，鉴定了包括AIF相互作用蛋白18和蛋白精氨酸甲基转移酶6(PRMT6)在内的13个HIT。在这里，我们关注AIF相互作用元件18，因为我们的初步数据表明，AIF相互作用元件18的敲除在鹦鹉中具有与PARP-1敲除一样的保护作用，并且它具有迄今未知的内切酶活性。因此，我们将AIF相互作用因子18命名为PAAN1(Parathatos依赖的AIF相关核酸酶1)。在指导的K99阶段，我们将首先确定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内切酶活性，并确定其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调节紧密包裹的基因组DNA中PAAN1的识别，从而在PARP-1激活后诱导染色质溶解和细胞死亡。PRMT6是一种组蛋白甲基转移酶，被认为参与了甲胎蛋白的死亡。在独立的R00期，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损伤部位的机制。我将使用两个高通量筛选进一步鉴定和鉴定甲胎蛋白所需的PAAN1相互作用蛋白的功能，以便了解PAAN1内切酶活性是如何受其网络调节的。该项目的成功完成将对PAAN1核酸内切酶活性和甲状旁腺激素的细胞控制产生重要的见解，并可能为开发治疗中风患者的创新治疗方法提供新的靶点。这种有指导的方法将在约翰·霍普金斯大学医学院的瓦利纳博士和泰德·道森博士以及雷蒙德·科勒博士的实验室进行。我可以使用Dawson博士和Koehler博士的实验室以及细胞工程研究所的神经再生和干细胞项目。为了成功地完成拟议的研究，在指导阶段，我打算参加表观遗传学课程，获得实验性中风模型和生物信息学技术方面的技能，以及赠款撰写和教学技能。这次培训将使我扩大我的专业知识，并帮助我转变为一名成功的独立学术研究人员。 公共卫生相关性：中风是世界上第二大致死和致残原因。中风或其他神经系统疾病后的死亡症是导致随后死亡、残疾和生活质量下降的主要原因。了解帕拉泰诺的潜在机制将为治疗中风或其他神经系统疾病的患者提供创新的治疗方法。