Identification of PKC delta Substrates in Stroke-reperfusion Injury

中风再灌注损伤中 PKC δ 底物的鉴定

基本信息

批准号：
7872305
负责人：
Wen-Hai Chou
金额：
$ 1.56万
依托单位：
ERNEST GALLO CLINIC AND RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-15 至 2009-11-28
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：中风是一种潜在的毁灭性神经系统疾病，发病率高和死亡率。溶栓剂（例如组织纤溶酶原激活剂（T-PA））当前是唯一可用于逆转急性缺血性中风的药物，但是循环的重新建立可能会自相矛盾地引发再灌注损伤。因此，有很大的兴趣开发治疗以限制再灌注损伤。最近对实验中风模型，小鼠遗传学和选择性肽抑制剂和活化剂的研究已涉及PKC4在中风 - 再灌注损伤中。特别是，在中性粒细胞中表达的PKC4似乎对于中风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鉴定PKC4在缺血和再灌注激活的信号通路中的唯一靶标。已经开发了一种新型的化学遗传学方法来鉴定激酶的直接底物。基于这种方法，我们产生了PKC4-Analog敏感突变体（PKC4-AS）酶，该酶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一种表达PKC4-AS的敲入小鼠。使用这些试剂，我们有独特的机会来鉴定PKC4的直接底物，以应对中风 - 再灌注损伤。该项目将首先描述PKC4-AS小鼠。在第二个特定目的中，我们将使用这种新型的化学基因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的中验证这些孤立的底物。这项工作将揭示在脑缺血期间激活的PKC4信号通路和中性粒细胞再灌注，并应促进与PKC4相关的治疗策略的发展。缺血性中风可能会被组织纤溶酶原激活剂（T-PA）逆转，但是血液流到缺血区域的恢复可以矛盾地产生再灌注损伤。许多证据支持蛋白激酶C三角洲在再灌注损伤中的关键作用。该建议是确定蛋白激酶C三角洲的靶标，并且获得的信息将促进开发新疗法以限制中风再灌注损伤。