Identification of PKC delta Substrates in Stroke-reperfusion Injury

描述（由申请人提供）：中风是一种潜在的破坏性神经系统疾病，具有高发病率和死亡率。溶栓剂如组织纤溶酶原激活剂（t-PA）是目前唯一可用于逆转急性缺血性卒中的药物，但血液循环的重建可能矛盾地引发再灌注损伤。因此，开发限制再灌注损伤的治疗方法非常有意义。最近对实验性脑卒中模型、小鼠遗传学和选择性肽抑制剂和激活剂的研究表明PKC4与脑卒中再灌注损伤有关。特别是，中性粒细胞中表达的PKC4似乎对卒中再灌注损伤至关重要。了解PKC4在再灌注损伤中的分子和细胞作用，需要确定PKC4在缺血和再灌注激活的信号通路中的独特靶点。一种新的化学遗传学方法已经开发出来，以确定激酶的直接底物。基于这种方法，我们已经产生了pkc4 -类似物敏感突变（PKC4-as）酶，它可以利用其他激酶不接受的特定ATP类似物，最近我们成功地产生了表达PKC4-as的敲入小鼠。有了这些试剂，我们有独特的机会鉴定PKC4在脑卒中再灌注损伤反应中的直接底物。该项目首先将pkc4作为小鼠进行表征。在第二个特定目标中，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PKC4底物。然后，我们将在第三个特定目标中验证这些分离的底物。这项工作将揭示中性粒细胞在脑缺血和再灌注过程中激活的PKC4信号通路，并促进PKC4相关治疗策略的发展。

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.