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，或者如果最初的应激是亚致死的，则对随后的热休克有抵抗力。热休克诱导细胞凋亡的机制和热休克蛋白对细胞凋亡的调控机制目前还不清楚。基于一种新兴的caspase激活范式和一种检测这种激活的新方法，我们发现热休克诱导的细胞凋亡是由一种涉及caspase-2的新途径启动的。相反，其他各种应激源，包括那些通过死亡受体连接、DNA损伤、内质网应激等起作用的应激源，不能参与caspase-2，而是通过caspase-8或caspase-9激活来启动细胞凋亡。我们的结果得到了caspase-2缺陷小鼠的初步结果的支持。我们将从这些考虑出发来处理这些目标。它们是：1；探讨热休克后caspase-2的活化及其在热休克诱导的细胞凋亡中的作用。我们将研究热休克和相关应激激活caspase-2的机制，以及这是如何导致细胞死亡的。我们的研究将采用新的方法来检测启动器caspase激活，基于新兴的caspase功能概念。我们将研究已知与caspase-2相互作用的蛋白质在热休克诱导的细胞凋亡中的可能作用，以及我们在这一过程中发现的与caspase-2相关的蛋白质。2. 研究热耐受性和热休克蛋白如何阻断热休克诱导的细胞凋亡。遭受亚致死热应激的细胞通过表达热休克蛋白，特别是HSP70，对随后的热休克产生抗性。我们将在细胞凋亡和caspase-2的激活方面研究这种耐药性，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。去除或下调热休克蛋白的影响将根据对caspase-2激活的潜在致敏性进行检查。3. 探讨热休克和caspase-2激活如何参与线粒体凋亡途径。Caspase-2并不直接处理刽子手caspase及其底物，因此热休克和Caspase-2活化如何杀死细胞并不明显。我们将探讨促凋亡Bcl-2家族成员及其线粒体通路在这一过程中的作用。特别是，我们将研究caspase-2介导的Bid切割如何与热应激对其他Bcl-2家族成员（Bax和Bak）的直接作用协同，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以热休克为代表的新型凋亡途径，并通过caspase-2的激活进行

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