Identification of PKC delta Substrates in Stroke-reperfusion Injury

描述（由申请人提供）：卒中是一种具有高发病率和死亡率的潜在破坏性神经系统疾病。组织纤溶酶原激活剂（t-PA）等溶栓药物是目前唯一可用于逆转急性缺血性卒中的药物，但重建循环可能矛盾地引发再灌注损伤。因此，人们对开发限制再灌注损伤的治疗方法非常感兴趣。最近的研究与实验性中风模型，小鼠遗传学，和选择性肽抑制剂和激活剂有牵连的PKC 4中风再灌注损伤。尤其是，在中性粒细胞中表达的PKC 4似乎是中风再灌注损伤所必需的。要了解PKC 4在再灌注损伤中的分子和细胞作用，需要确定缺血和再灌注激活的信号通路中PKC 4的独特靶点。一种新的化学遗传学方法已经开发出来，以确定直接底物的激酶。基于这种方法，我们已经产生了一个PKC 4类似物敏感突变体（PKC 4-as）酶，可以利用特定的ATP类似物，不接受其他激酶，最近我们成功地产生了一个敲入小鼠表达PKC 4-as。有了这些试剂，我们有独特的机会，以确定直接底物的PKC 4在中风再灌注损伤。该项目将首先表征PKC 4-作为小鼠。在第二个具体目标中，我们将使用这种新的化学遗传学方法来标记和鉴定中性粒细胞中的PKC 4底物。然后，我们将在第三个具体目标中验证这些分离的底物。这项工作将揭示PKC 4信号通路激活脑缺血和再灌注过程中的中性粒细胞，并应促进PKC 4相关的治疗策略的发展。 组织纤溶酶原激活剂（t-PA）可逆转缺血性卒中，但缺血区血流恢复可产生再灌注损伤。许多证据支持蛋白激酶C δ在再灌注损伤中的关键作用。该提案旨在确定蛋白激酶C δ的靶点，所获得的信息将促进限制中风再灌注损伤的新疗法的开发。

DESCRIPTION (provided by applicant): Stroke is a potentially devastating neurological disorder with high morbidity and mortality. Thrombolytic agents such as tissue plasminogen activator (t-PA) are currently the only drugs available to reverse acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Therefore, there is great interest in developing treatments to limit reperfusion injury. Recent studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have implicated PKC4 in stroke-reperfusion injury. In particular, PKC4 expressed in neutrophils appears essential for stroke- reperfusion injury. Understanding the molecular and cellular actions of PKC4 in reperfusion injury requires the identification of the unique targets of PKC4 in signaling pathways activated by ischemia and reperfusion. A novel chemical-genetics approach has been developed to identify immediate substrates of kinases. Based on this approach, we have generated a PKC4-analog sensitive mutant (PKC4-as) enzyme that can utilize specific ATP analogs that are not accepted by other kinases, and recently we succeeded in generating a knock-in mouse expressing PKC4-as. With these reagents, we have the unique opportunity to identify direct substrates of PKC4 in response to stroke-reperfusion injury. This project will first characterize the PKC4-as mice. In the second specific aim, we will use this novel chemical-genetics approach to label and identify PKC4 substrates in neutrophils. We will then validate these isolated substrates in the third specific aim. This work will reveal PKC4 signaling pathways activated during cerebral ischemia and reperfusion in neutrophils, and should facilitate the development of PKC4-related therapeutic strategies. Ischemic stroke can potentially be reversed by tissue plasminogen activator (t-PA), but the restoration of blood flow to ischemic areas can paradoxically produce reperfusion injury. Much evidence supports a key role of protein kinase C delta in reperfusion injury. This proposal is to identify the targets of protein kinase C delta, and the information obtained will facilitate the development of new therapies to limit stroke- reperfusion injury.