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法清除闭塞，以及缺血心脏的再灌流。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，人们对确定损伤的分子基础以及开发预防或减少脑梗塞的方法非常感兴趣。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，另一种涉及尝试通过引入干细胞来修复损伤。目前的应用坚持这两种方法都具有同等重要性的论点。对包括啮齿动物、兔、狗和猪在内的多种物种的研究表明，当细胞凋亡受到抑制时，缺血/再灌注引起的脑梗塞面积可以减少50%或更多。多种心脏保护策略在BH3-唯一促凋亡蛋白的水平上汇聚，在那里做出不可逆转的死亡/生存决定。这些蛋白质代表了抗细胞凋亡治疗的最终前沿，并提供了高度特异性的靶点。Bnip3已经成为缺血和再灌注过程中细胞死亡的中心调节因子，但我们几乎不知道它的作用机制，它是如何被激活的，或者它是如何被最有效地抑制的。我们在这里提出了两个主要假设：第一，Bnip3是一种需要依赖PKC？的步骤才能激活的磷酸蛋白；第二，Bnip3通过与亲环素-D相互作用，独立于其他仅依赖BH3的蛋白激活线粒体通透性转换孔。本申请有5个目的：(1和2)明确磷酸化在Bnip3分子调控中的作用，并确定该激酶(S)。(3)确定Bnip3在单纯缺血和缺血再灌流条件下的死亡途径。(4)确定Bnip3在促进脑梗塞的其他BHS-Only蛋白(Bid、Bad、PUMA、noxa)中的关系和功能层次。(5)确定Bnip3 N-末端的一个功能区，该功能区在跨膜结构域缺失时具有心脏保护作用。这些目标的成功实现将为Bnip3在心肌梗死中的作用提供更全面的表征，并测试治疗再灌注损伤的新的分子方法。

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.