Role of Protein Phosphatase-1 in Cerebral Ischemia and Cell Death

蛋白磷酸酶 1 在脑缺血和细胞死亡中的作用

• 批准号: 8016671
• 负责人: HUGH C HEMMINGS
• 金额: $ 36.02万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8016671
• 关键词: Address; Apoptosis; Apoptotic; Attention; Attenuated; Brain; Calcium/calmodulin-dependent protein kinase; Canis familiaris; Catalytic Domain; Cell Culture Techniques; Cell Death; Cell Survival; Cell physiology; Cells; Cerebral Ischemia; Cessation of life; Clinical; Co-Immunoprecipitations; Complex; Development; Enzymes; Family suidae; Foundations; Glucose; Glutamate Receptor; Glutamates; Goals; Health; Healthcare; Heart Arrest; Hippocampus (Brain); Holoenzymes; Hour; Hydrogen Peroxide; Impairment; In Vitro; Ischemia; Lead; Link; Mass Fragmentography; Mass Spectrum Analysis; Mediator of activation protein; Medical; Medical Economics; Membrane; Modeling; Molecular; N-Methylaspartate; Na(+)-K(+)-Exchanging ATPase; Necrosis; Nervous System Trauma; Neurologic; Neurons; Nitric Oxide Synthase; Nitrogen; Oxygen; Pathway interactions; Peptide Hydrolases; Phosphorylation; Physiological; Play; Process; Property; Prosencephalon; Protein Kinase; Protein Kinase C; Protein Phosphatase Inhibitor; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Proteomics; Reagent; Regulation; Reperfusion Therapy; Research Proposals; Resuscitation; Role; Scaffolding Protein; Second Messenger Systems; Shotguns; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Simulate; Site; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Testing; Translations; base; brain cell; calmodulin-dependent protein kinase II; cell growth regulation; cell injury; deprivation; design; economic impact; excitotoxicity; genetic regulatory protein; in vivo; inhibitor/antagonist; member; neuroblastoma cell; novel; prevent; protein phosphatase inhibitor-1; reconstitution; research study; response; second messenger; small molecule; social; tandem mass spectrometry; therapeutic target; voltage

DESCRIPTION (provided by applicant): Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite this major clinical and economic impact, there is currently no specific medical therapy. Global cerebral ischemia is associated with extensive necrotic and apoptotic cell death; these processes are tightly regulated by several mechanisms, including a critical role for protein phosphorylation. While the involvement of protein kinases in the control of cell death in global cerebral ischemia is well established, the role of protein phosphatases has received relatively little attention. Protein phosphatase-1 is a member of the serine/threonine protein phosphatase subfamily that has been implicated in the regulation of cell death. We have identified and purified several novel multimeric forms of protein phosphatase-1 in mammalian brain. The activity of one of these, termed protein phosphatase-1IC (PP-1IC), is activated in vivo in pig and dog models of global cerebral ischemia as well as in cell culture models of ischemia. Based on these novel findings, we hypothesize that PP-1IC is a component of the signal transduction pathways that link global cerebral ischemia to cell death. In order to determine the mechanisms of activation and the role of PP-1IC in global cerebral ischemia, we propose to purify and characterize the PP-1IC holoenzyme from control and ischemic pig forebrain following cardiac arrest with resuscitation and reperfusion. The molecular compositions of native PP-1IC purified from control and ischemic brain will be determined by mass spectrometry, and the mechanisms of PP-1IC regulation will be studied by reconstitution of the identified components in vitro. Specific membrane permeable inhibitors of PP-1 and other reagents will be developed and used to determine the role of PP-1IC as a mediator of cell death in cell culture models of cerebral ischemia. The mechanisms of ischemic activation of PP-1IC will be investigated based on the hypothesis that the Ca2+regulated protein kinases, Ca2????dependent protein kinase II (CaMKII) and/or protein kinase C4 (PKC4) function as upstream regulators. These studies will elucidate physiological and pathophysiological mechanisms that regulate native PP-1 holoenzyme activity in brain and define the role of PP-1 in the control of cell death in global cerebral ischemia. This functional proteomics approach is targeted to the development of rational mechanism-based therapies to attenuate ischemic brain cell death, with a long-term goal of clinical translation. PUBLIC HEALTH RELEVANCE: Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite an immense social, medical and economic impact, there is currently no specific pharmacological therapy. The proposed studies will elucidate physiological and pathophysiological mechanisms that regulate native protein phosphatase-1 holoenzymes in brain and determine their role in the control of cell death in global cerebral ischemia. This approach is targeted to the development of rational mechanism-based protein phosphatase-1 inhibitors to attenuate ischemic cell death and neurological injury.

描述（由申请人提供）：由于心脏骤停导致的全身脑缺血会导致神经系统衰弱，需要昂贵的长期医疗保健。尽管有这种重大的临床和经济影响，但目前还没有具体的药物治疗方法。全脑缺血与广泛的坏死和凋亡细胞死亡有关；这些过程受到多种机制的严格调控，其中包括蛋白质磷酸化的关键作用。虽然蛋白激酶参与控制全脑缺血细胞死亡已得到充分证实，但蛋白磷酸酶的作用相对较少受到关注。蛋白磷酸酶-1 是丝氨酸/苏氨酸蛋白磷酸酶亚家族的成员，与细胞死亡的调节有关。我们在哺乳动物大脑中鉴定并纯化了几种新型多聚体形式的蛋白磷酸酶-1。其中一种称为蛋白磷酸酶-1IC (PP-1IC) 的活性在猪和狗的全脑缺血模型以及缺血的细胞培养模型中体内被激活。基于这些新发现，我们假设 PP-1IC 是连接全脑缺血与细胞死亡的信号转导途径的一个组成部分。为了确定 PP-1IC 在全脑缺血中的激活机制和作用，我们建议从心脏骤停伴复苏和再灌注后的对照猪前脑和缺血猪前脑中纯化和表征 PP-1IC 全酶。从对照和缺血脑中纯化的天然PP-1IC的分子组成将通过质谱测定，并且PP-1IC的调节机制将通过在体外重构所鉴定的成分来研究。将开发 PP-1 的特异性膜渗透抑制剂和其他试剂，并用于确定 PP-1IC 作为脑缺血细胞培养模型中细胞死亡介质的作用。基于Ca2+调节蛋白激酶、Ca2+依赖性蛋白激酶II(CaMKII)和/或蛋白激酶C4(PKC4)作为上游调节剂的假设，将研究PP-1IC的缺血激活机制。这些研究将阐明调节大脑中天然 PP-1 全酶活性的生理和病理生理机制，并确定 PP-1 在控制全脑缺血中细胞死亡中的作用。这种功能蛋白质组学方法的目标是开发基于合理机制的疗法，以减轻缺血性脑细胞死亡，并以临床转化为长期目标。公共健康相关性：心脏骤停导致的全脑缺血会导致神经功能损伤，导致需要昂贵的长期医疗保健。尽管具有巨大的社会、医学和经济影响，但目前尚无具体的药物治疗方法。拟议的研究将阐明调节大脑中天然蛋白磷酸酶-1全酶的生理和病理生理机制，并确定它们在控制全脑缺血中细胞死亡中的作用。该方法旨在开发基于合理机制的蛋白磷酸酶 1 抑制剂，以减轻缺血性细胞死亡和神经损伤。