Mechanisms of action and activation of the death-inducing protein Bnip3

描述（由申请人提供）：据估计，美国每年有150万例急性冠状动脉事件。大多数是由冠状动脉闭塞引起的，40%是致命的。治疗通常包括机械或酶去除闭塞，再灌注缺血心脏。一段时间以来，人们已经认识到再灌注可促进额外的心脏损伤和细胞死亡，从而加剧梗死的过程。因此，人们对识别损伤的分子基础和开发预防或减少梗死的方法非常感兴趣。作为治疗心肌梗死的一种手段，有两种方法正在受到严格的审查，一种是通过心脏保护来减少再灌注时的损伤，另一种是通过引入干细胞来修复损伤。目前的应用坚持这两种方法同等重要的论点。对啮齿类动物、兔子、狗和猪等多种动物的研究表明，当细胞凋亡受到抑制时，缺血/再灌注引起的梗死面积可减少50%或更多。多种心脏保护策略在仅bh3促凋亡蛋白水平汇聚，在此水平上做出不可逆转的死亡/生存决定。这些蛋白代表了抗细胞凋亡治疗的最后前沿，并提供了高特异性的靶点。Bnip3已成为缺血和再灌注期间细胞死亡的中心调节因子，但我们对其作用机制、如何激活或如何最有效地抑制它几乎一无所知。我们在此提出验证两个主要假设：首先，Bnip3是一种需要PKC-？-依赖激活步骤；其次，Bnip3通过与cyclophillin-D相互作用，独立于其他BH3-only蛋白激活线粒体通透性过渡孔。该申请有5个目的：（1 & 2）确定磷酸化在Bnip3分子调控中的作用，并鉴定激酶。(3)探讨单独缺血和缺血再灌注条件下bnip3介导的死亡途径。(4)明确Bnip3与其他BHS-only蛋白（Bid, Bad， PUMA， NOXA）之间的关系和功能层次。(5)确定Bnip3 n端在跨膜结构域被删除时具有心脏保护作用的功能区域。这些目标的成功实现将为Bnip3在心肌梗死中的作用提供更完整的表征，并为再灌注损伤的治疗提供新的分子方法。

DESCRIPTION (provided by applicant): There are an estimated 1.5 million cases of acute coronary events in the U.S. annually. Most are caused by occlusion of coronary arteries and 40% are fatal. Treatment usually involves mechanical or enzymatic removal of the occlusion, and reperfusion of the ischemic heart. It has been recognized for some time that reperfusion promotes additional cardiac injury and cell death that exacerbates the process of infarction. As a consequence, there is great interest in identifying the molecular basis of the damage and developing methods to prevent or reduce infarction. Two approaches are under intense scrutiny as a means to treat myocardial infarction, one involves cardioprotection to reduce injury at the time of reperfusion, and the other involves attempts to repair the damage by introducing stem cells. The current application adheres to the thesis that both of these approaches are of equivalent importance. Studies in multiple species including rodents, rabbits, dogs, and pigs have demonstrated that the size of an infarct caused by ischemia/reperfusion is reduced 50% or more when apoptosis is inhibited. Multiple cardioprotective strategies converge at the level of the BH3-only pro-apoptotic proteins, where the irreversible death/survival decision is made. These proteins represent the final frontier for anti-apoptosis therapy and offer targets of high specificity. Bnip3 has emerged as a central regulator of cell death during both ischemia and reperfusion, yet we know virtually nothing about its mechanism of action, how it is activated, or how it may be most effectively inhibited. We propose here to test 2 main hypotheses: firstly, Bnip3 is a phosphoprotein that requires a PKC-?-dependent step for activation; secondly, Bnip3 activates the mitochondrial permeability transition pore independently of other BH3-only proteins by interacting with cyclophillin-D. The application has 5 aims: (1 & 2) To define the roles of phosphorylation in the molecular regulation of Bnip3 and identify the kinase(s). (3) To determine the pathway of Bnip3-mediated death under conditions of ischemia alone and ischemia with reperfusion. (4) Define the relationships and functional hierarchy of Bnip3 within other BHS-only proteins (Bid, Bad, PUMA, NOXA) that are implicated in promoting infarction. (5) Define a functional region of the Bnip3 N-terminus that is cardioprotective when the transmembrane domain is deleted. Successful achievement of these aims will provide a more complete characterization of the role of Bnip3 in myocardial infarction and test a new molecular approach to therapy for reperfusion injury.