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

描述（由申请人提供）：美国每年估计有150万例急性冠状动脉事件。大多数是由冠状动脉阻塞引起的，40%是致命的。治疗通常涉及机械或酶清除闭塞，以及缺血心脏的再灌注。一段时间以来，人们已经认识到，再灌注促进了额外的心脏损伤和细胞死亡，从而加剧了梗死的过程。因此，人们对鉴定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。两种方法作为治疗心肌梗死的手段受到严格审查，一种涉及心脏保护以减少再灌注时的损伤，另一种涉及通过引入干细胞来修复损伤的尝试。本申请坚持这两种方法具有同等重要性的论点。在包括啮齿动物、兔、狗和猪在内的多个物种中的研究已经证明，当细胞凋亡被抑制时，由缺血/再灌注引起的梗死的大小减少50%或更多。多种心脏保护策略在仅BH 3促凋亡蛋白的水平上汇聚，其中做出不可逆的死亡/存活决定。这些蛋白质代表了抗凋亡治疗的最后前沿，并提供了高特异性的靶标。bnip 3已经成为缺血和再灌注过程中细胞死亡的中心调节因子，但我们对它的作用机制、如何激活或如何最有效地抑制它几乎一无所知。我们建议在这里测试2个主要假设：首先，Bnip 3是一种磷蛋白，需要PKC-？-激活的依赖步骤;第二，Bnip 3通过与亲环蛋白-D相互作用而独立于其他仅含BH 3的蛋白激活线粒体通透性转换孔。本申请具有5个目的：（1和2）确定磷酸化在Bnip 3的分子调节中的作用并鉴定激酶。(3)确定在单独缺血和缺血再灌注条件下Bnip 3介导的死亡途径。(4)定义Bnip 3在其他促进梗死的仅BHS蛋白（Bid，Bad，BRA，NOXA）中的关系和功能层次。(5)定义Bnip 3 N-末端的功能区域，当跨膜结构域缺失时，该功能区域具有心脏保护作用。这些目标的成功实现将为Bnip 3在心肌梗死中的作用提供更完整的表征，并测试再灌注损伤治疗的新分子方法。

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.