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

描述(申请人提供)：由于心脏骤停导致的全球脑缺血导致衰弱的神经损伤，需要昂贵的长期医疗护理。尽管有这种重大的临床和经济影响，但目前还没有专门的药物治疗。全脑缺血与广泛的坏死性和凋亡性细胞死亡有关；这些过程受到几种机制的严格调控，包括蛋白质磷酸化的关键作用。虽然蛋白激酶在控制全脑缺血细胞死亡中的作用已得到充分证实，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中的一员，参与细胞死亡的调节。我们已经在哺乳动物脑中鉴定和纯化了几种新的多聚体蛋白磷酸酶-1。其中一种被称为蛋白磷酸酶-1IC(PP-1IC)的活性在猪和狗的全脑缺血模型以及细胞培养的缺血模型中被激活。基于这些新的发现，我们假设PP-1IC是将全脑缺血与细胞死亡联系起来的信号转导通路的组成部分。为了探讨PP-1IC的激活机制及其在全脑缺血中的作用，我们从正常和缺血的猪前脑中分离纯化PP-1IC全酶，并对其进行鉴定。从正常和缺血脑中分离纯化的天然PP-1IC将用质谱仪测定其分子组成，并通过在体外重组已鉴定的成分来研究PP-1IC的调控机制。将开发PP-1和其他试剂的特异膜通透性抑制剂，并用于确定PP-1IC在脑缺血细胞培养模型中作为细胞死亡介质的作用。基于钙离子调节的蛋白激酶、钙依赖性蛋白激酶II(CaMKII)和/或蛋白激酶C4(PKC4)作为上游调节因子的假说，将探讨PP-1IC缺血激活的机制。这些研究将阐明调节脑内天然PP-1全酶活性的生理和病理生理学机制，并明确PP-1在控制全脑缺血时细胞死亡中的作用。这种功能蛋白质组学方法的目标是开发合理的基于机制的疗法来减轻缺血性脑细胞死亡，并有一个长期的临床转化目标。

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.