Heat Shock-Induced Apoptosis

描述(申请人提供)：对热应激的反应是古老的和高度保守的。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，则对随后的热休克产生抵抗。热休克诱导细胞凋亡的机制以及热休克蛋白对细胞凋亡的调控机制还不是很清楚。基于一种新兴的启动子caspase激活模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涉及caspase-2的新途径启动的。相反，各种其他应激源，包括通过死亡受体连接、DNA损伤、内质网应激等方式发挥作用的应激因素，都不能激活caspase-2，而是通过激活caspase-8或caspase-9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。从这些考虑出发，我们将解决的目标如下。1.探讨热休克后caspase-2的激活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压力激活caspase-2的机制，以及这是如何导致细胞死亡的。我们的研究将基于caspase功能的新概念，采用新的方法来检测启动子caspase的激活。已知的与caspase-2相互作用的蛋白质将被研究它们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.确定耐热性和热休克蛋白在阻断热休克诱导的细胞凋亡中的作用。受到亚致死热应激的细胞通过表达热休克蛋白，特别是热休克蛋白70，对随后的热休克产生抵抗力。我们将从细胞凋亡和caspase-2的激活两个方面来研究这种抗性，并将其与其他机制进行比较，如抗凋亡的Bcl-2家族成员的表达。移除或下调HSPs的效果将从对caspase-2激活的潜在敏感度方面进行检测。3.探讨热休克和半胱氨酸天冬氨酸氨基转移酶-2(caspase-2)激活与线粒体凋亡途径的关系。Caspase-2不直接处理执行者caspase或其底物，因此热休克和caspase-2激活如何杀死细胞并不明显。我们将探讨促凋亡的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caspase-2介导的Bid裂解如何与热应激对其他Bcl-2家族成员(Bax和Bak)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途径来阐明以热休克为代表的新的凋亡途径，并通过激活caspase-2来进行。

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