Oxidants & Caspases: Initiation of Reperfusion Injury

描述（由申请人提供）：半胱天蛋白酶或其调节因子的治疗性操作在控制缺血/再灌注中的细胞损伤方面具有重要的前景。然而，由于阻断特定的半胱天冬酶可通过替代或代偿途径导致细胞凋亡进展，因此了解单个半胱天冬酶和那些直接控制半胱天冬酶活性或提供“短路”导致所谓的“不依赖半胱天冬酶”的下游细胞死亡机制的分子的作用至关重要。该建议的中心假设是心肌细胞缺血/再灌注（I/R）后，活性氧（ROS）的产生和caspase-2的激活导致线粒体扰动，导致细胞色素c （cyt c）的释放和电子传递链的破坏和凋亡细胞死亡。通过结合细胞和动物模型，通过RNA干扰和特异性敲除小鼠，我们提出研究特异性caspase及其调节因子（包括Smac/DIABLO和凋亡抑制蛋白（IAPs））在两个主要特定目的中的作用：1)确定caspase-2、线粒体ROS、Bax和Bid在缺血/再灌注诱导心肌细胞损伤中的相互作用。我们假设IR导致线粒体ROS的产生以及Bax和caspase-2的激活。在这些研究中，我们还将研究特定ROS，如超氧化物和过氧化氢，以及抗氧化处理对Bax和caspase-2活化的影响。此外，我们假设活性caspase-2直接作用于线粒体和/或通过Bid切割导致cyt c的释放和进一步的ROS生成；2)确定caspase-2在促凋亡线粒体蛋白Smac/DIABLO释放中的作用，其对IAP阻断caspase活性的影响，以及它们在I/ r诱导细胞凋亡扩增中的相对作用。我们假设，在I/ r刺激的心肌细胞中，caspase可以引起凋亡线粒体蛋白的差异释放，如cyt c和抑制iap的Smac/DIABLO，并且这些影响caspase介导的I/ r诱导的细胞死亡扩增。更好地了解特定的半胱天冬酶活性以及半胱天冬酶、线粒体ROS和内源性调节因子之间的生理相互作用，对于评估这些靶点的治疗潜力至关重要。

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.