Oxidants & Caspases: Initiation of Reperfusion Injury

描述（由申请人提供）：半胱天冬酶或其调节剂的治疗操作在控制缺血/再灌注中的细胞损伤方面提供了重要的前景。由于特定半胱天冬酶的阻断可以通过替代或补偿途径导致细胞凋亡进展，然而，理解单个半胱天冬酶和直接控制半胱天冬酶活性或提供导致所谓的“非半胱天冬酶”下游细胞死亡机制的“短路”的那些分子的作用是至关重要的。该建议的中心假设是，在心肌细胞中的缺血/再灌注（I/R）之后，活性氧（ROS）的产生和半胱天冬酶-2活化导致线粒体扰动，从而导致细胞色素c（cyt c）的释放和电子传递链的破坏以及凋亡性细胞死亡。使用细胞和动物模型的组合，通过RNA干扰和特定敲除小鼠消除特定蛋白，我们建议研究特定半胱天冬酶及其调节剂的作用，包括Smac/DIABLO和凋亡蛋白抑制剂（IAP）在两个主要特定目标中的作用：1）探讨caspase-2、线粒体ROS、Bax和Bid在心肌缺血/再灌注损伤中的相互作用。我们推测IR导致线粒体ROS的产生以及Bax和caspase-2的活化。在这些研究中，我们还将研究特定的ROS，如超氧化物和过氧化氢，以及抗氧化剂处理对Bax和caspase-2激活的影响。进一步，我们推测，活性caspase-2直接作用于线粒体和/或通过Bid切割，导致细胞色素c的释放和进一步的ROS产生; 2）确定caspase-2在促凋亡线粒体蛋白Smac/DIABLO的释放中的作用，其对IAP阻断caspase活性的影响以及它们在I/R诱导的细胞凋亡放大中的相对作用。我们推测，在I/R刺激的心肌细胞，半胱天冬酶可以导致差异释放的促凋亡线粒体蛋白，如细胞色素c和IAP抑制Smac/DIABLO，这些影响半胱天冬酶介导的放大I/R诱导的细胞死亡。更好地了解特定的caspase活性和caspase，线粒体ROS和作用于线粒体或从线粒体释放的内源性调节剂之间的生理相互作用将是评估这些目标的治疗潜力的关键。

DESCRIPTION (provided by applicant): Therapeutic manipulation of caspases or their regulators offers significant promise in controlling cellular injury in ischemia/reperfusion. Because blockade of specific caspases can result in apoptotic progression via alternative or compensatory pathways, however, it is critical to understand the roles of individual caspases and those molecules which directly control caspase activities or which provide "short-circuits" leading to so-called "caspase independent" downstream mechanisms of cell death. The central hypothesis of this proposal is that following ischemia/reperfusion (I/R) in cardiomyocytes, generation of reactive oxygen species (ROS) and caspase-2 activation lead to mitochondrial perturbations resulting in release of cytochrome c (cyt c) and disruption of the electron transport chain and apoptotic cell death. Using a combination of cellular and animal models, depletion of specific proteins via RNA interference and specific knockout mice, we propose to study the roles of specific caspases and their regulators, including Smac/DIABLO and inhibitor of apoptosis proteins (IAPs) in two major specific aims: 1) Determine the interactive roles of caspase-2, mitochondrial ROS, and Bax and Bid ininitiating ischemia/reperfusion-induced injury to cardiomyocytes. We hypothesize that IR results in generation of mitochondrial ROS and the activation of Bax and caspase-2. In these studies we will examine also the effects of specific ROS, such as superoxide and hydrogen peroxide, and antioxidant treatment on the activation of Bax and caspase-2. Further, we postulate that active caspase-2 acts directly on the mitochondria and/or via Bid cleavage to cause the release of cyt c and further ROS generation; 2) Determine the role of caspase-2 in the release of pro-apoptotic mitochiondrial protein Smac/DIABLO, its effects on IAP blockade of caspase activity and their relative roles in amplification of I/R-induced apoptosis. We hypothesize that in I/Rstimulated cardiomyocytes, caspases can cause differential release of apoptogenic mitochondrial proteins, such as cyt c and the IAP-inhibiting Smac/DIABLO, and that these affect caspase-mediated amplification of I/R-induced cell death. A better understanding of specific caspase activity and the physiological interplay between caspases, mitochondrial ROS, and the endogenous regulators which act on or are released from the mitochondria will be critical in assessing the therapeutic potential of these targets.