Heat Shock-Induced Apoptosis

描述（由申请人提供）：对热应激的反应是古老且高度保守的。在动物中，它经常导致细胞凋亡，或者如果初始应激是亚致死的，则导致对随后的热休克的抵抗。热休克诱导细胞凋亡的机制及其受热休克蛋白调控的机制尚不清楚。基于一个新兴的范例的启动caspase激活和一种新的方法来检测这种激活，我们已经发现，热休克诱导的细胞凋亡是由一个新的途径，涉及caspase-2。相比之下，各种其他应激源，包括通过死亡受体连接、DNA损伤、内质网应激等起作用的那些，不能参与半胱天冬酶-2，而是通过半胱天冬酶-8或半胱天冬酶-9激活启动细胞凋亡。我们的研究结果得到了caspase-2缺陷小鼠的初步结果的支持。我们将讨论的目标来自这些考虑。 这些是：1.探讨热休克后caspase-2的活化及其在热休克诱导细胞凋亡中的作用。我们将研究热休克和相关应激激活caspase-2的机制，以及这如何导致细胞死亡。我们的研究将采用新的方法来检测启动caspase激活，基于caspase功能的新兴概念。已知与caspase-2相互作用的蛋白质将被检查其在热休克诱导的细胞凋亡中的可能作用，我们在此过程中发现的与caspase-2相关的蛋白质也将被检查。2.探讨耐热蛋白和热休克蛋白如何阻断热休克诱导的细胞凋亡。受到亚致死热应激的细胞通过表达热休克蛋白，特别是HSP 70，变得对随后的热休克具有抗性。我们将研究这种抵抗与细胞凋亡和caspase-2的激活，并将其与其他机制，如抗凋亡Bcl-2家族成员的表达进行比较。去除或下调HSP的作用将在对半胱天冬酶-2活化的潜在敏化方面进行检查。3.探讨热休克和caspase-2激活如何参与线粒体凋亡途径。半胱天冬酶-2不直接处理执行者半胱天冬酶或其底物，因此热休克和半胱天冬酶-2活化如何杀死细胞并不明显。我们将探讨促凋亡Bcl-2家族成员和线粒体途径在这一过程中的作用。特别是，我们将研究如何caspase-2介导的Bid裂解可能与热应激对其他Bcl-2家族成员（Bax和巴克）的直接影响协同作用，通过线粒体途径促进细胞凋亡。这三个目标代表了独立和互补的方法来阐明新的凋亡途径所代表的热休克和进行通过激活半胱天冬酶-2

DESCRIPTION (provided by applicant): The response to heat stress is ancient and highly conserved. In the animals it frequently results in apoptosis, or if the initial stress is sub-lethal, resistance to subsequent heat shock. While readily demonstrated, the mechanisms of heat shock-induced apoptosis and its regulation by heat shock proteins are not well understood. Based on an emerging paradigm of initiator caspase activation and a novel method to detect such activation, we have found that heat shock-induced apoptosis is initiated by a novel pathway involving caspase-2. In contrast, a variety of other stressors, including those working by death receptor ligation, DNA damage, endoplasmic reticulum stress, and others, fail to engage caspase-2, and instead initiate apoptosis via either caspase-8 or caspase-9 activation. Our results are supported by preliminary results from caspase-2-deficient mice. The aims we will address follow from these considerations. These are: 1. To investigate the activation of caspase-2 following heat shock and its role in heat shock-induced apoptosis. We will investigate the mechanisms involved in the activation of caspase-2 by heat shock and related stress, and how this leads to the death of the cell. Our studies will employ new approaches to the detection of initiator caspase activation, based on emerging concepts of caspase function. Proteins known to interact with caspase-2 will be examined for their possible roles in heat shock-induced apoptosis, as will proteins we find in association with caspase-2 during this process. 2. To determine how thermotolerance and heat shock-proteins function to block heat shock-induced apoptosis. Cells that are subjected to sub-lethal heat stress become resistant to subsequent heat shock, through the expression of heat shock proteins, especially HSP70. We will examine this resistance with respect to apoptosis and the activation of caspase-2, and compare it with other mechanisms such as expression of the anti-apoptotic Bcl-2 family members. Effects of removing or down-regulating HSPs will be examined in terms of potential sensitization to caspase-2 activation. 3. To explore how heat shock and caspase-2 activation engage the mitochondrial pathway of apoptosis. Caspase-2 does not directly process executioner caspases or their substrates, and therefore how heat shock and caspase-2 activation kill cells is not obvious. We will probe the role of pro-apoptotic Bcl-2 family members and the mitochondrial pathway in this process. In particular, we will investigate how caspase-2-mediated cleavage of Bid may synergize with direct effects of heat stress on other Bcl-2 family members (Bax and Bak) to promote apoptosis via the mitochondrial pathway. These three goals represent independent and complimentary approaches to the elucidation of the novel apoptotic pathway represented by heat shock and proceeding through the activation of caspase-2