Signaling Cell Death from the Endoplasmic Reticulum

描述(由申请人提供)：癌细胞必须避开凋亡检查点才能在不利的条件下生存，如营养缺乏和缺氧，这会迅速导致对内质网(ER)蛋白质折叠能力的过度需求。当内质网管腔中未折叠蛋白的范围达到临界水平时，细胞参与一系列进化上保守的信号转导途径，统称为未折叠蛋白反应(UPR)。哺乳动物细胞中UPR的主要效应者是内质网驻留的跨膜传感器IRE11、PERK和AFT6。这些压力传感器最初扩展内质网，上调伴侣并阻止全球翻译，以恢复内环境平衡。然而，如果内质网损伤严重，这些内质网驻留应激传感器通过鲜为人知的机制启动细胞凋亡。持续和高水平的内质网应激在许多形式的癌症中都有记录；因此，恶性细胞必须进化机制来逃避这种应激的正常细胞毒性后果。恢复UPR的凋亡产物的努力有望成为杀死癌细胞的治疗策略。过度的内质网应激触发“内在”的凋亡途径，该途径在线粒体膜外受到促死亡的bcl2家族蛋白Bax和BAK的严密调控。然而，从内质网应激到线粒体Bax/BAK激活的分子链仍然知之甚少。我的实验室已经开发了一种从内质网应激的Bax-/-Bak-/-细胞的胞浆提取物中纯化线粒体前凋亡活性的方法。利用这项技术，我们已经确定了两个主要的凋亡信号，它们汇聚在线粒体Bax/BAK上。一个信号是BH3-Only蛋白BID，它被Caspase-2切割成更短的促凋亡形式。我们现在试图了解导致从内质网膜错误折叠蛋白的感知到Caspase-2的催化激活的事件，Caspase-2是哺乳动物中特征最差的Caspase之一。从活性提取物中，我们最近还纯化了第二个新的成分-一个包含一个SH2结构域和两个SH3结构域的接头蛋白，我们的数据表明这是一个在内质网应激下游的Bid非依赖性的凋亡信号。我们现在的目标是确定这个接头蛋白在内质网应激信号中的促凋亡作用。这项建议的长期目标是了解细胞如何检测内质网应激，确定损伤是否致命，并将这些信息传达给细胞死亡机制，并确定可以操纵的途径中可以影响细胞存活的成分。两个特定的目标是：(1)确定内质网应激激活Caspase-2的机制(S)；(2)确定这个含有SH2/SH3结构的接头蛋白在内质网应激信号中的作用。这些研究将确定内质网应激下游控制细胞凋亡的机制--这一途径可能代表癌细胞的关键治疗靶点。

DESCRIPTION (provided by applicant): Cancer cells must avert apoptotic checkpoints to survive in unfavorable conditions, such as nutrient deprivation and hypoxia, which quickly lead to excessive demand on the protein folding capacity of the endoplasmic reticulum (ER). When the extent of unfolded proteins in the ER lumen reaches a critical level, the cell engages a set of evolutionarily conserved signal transduction pathways that are collectively known as the unfolded protein response (UPR). Major effectors of the UPR in mammalian cells are the ER-resident transmembrane sensors IRE11, PERK, and AFT6. These stress sensors initially expand the ER network, upregulate chaperones and arrest global translation to restore homeostasis. However, if the ER damage is severe, these ER resident stress sensors initiate apoptosis through poorly understood mechanisms. Sustained and high level ER stress is documented in many forms of cancer; hence, malignant cells must evolve mechanisms to evade the normally cytotoxic consequences of such stress. Efforts to restore the apoptotic outputs of the UPR hold promise as a therapeutic strategy to kill cancer cells. Excessive ER stress triggers the "intrinsic" apoptotic pathway, which is tightly regulated at the outer mitochondrial membrane by the pro-death BCL-2 family proteins BAX and BAK. However, the molecular chain of events leading from ER stress to mitochondrial BAX/BAK activation remains poorly understood. My laboratory has developed a process to purify the pre- mitochondrial apoptotic activity from the cytosolic extract of ER-stressed Bax-/-Bak-/- cells. Using this technology, we have identified two major apoptotic signals that converge on mitochondrial BAX/BAK. One signal is the BH3-only protein BID, which is cleaved into its shorter pro-apoptotic form by Caspase-2. We now seek to understand the events that lead from the sensing of misfolding proteins at the ER membrane to the catalytic activation of Caspase-2, one of the most poorly characterized mammalian caspases. From the active extract, we also recently purified a second novel component-an adaptor protein containing an SH2 domain and two SH3 domains, which our data suggest is a BID-independent apoptotic signal downstream of ER stress. We now aim to define the pro-apoptotic role of this adaptor protein in ER stress signaling. The long- term objectives of this proposal are to understand how cells detect ER stress, decide if the damage is lethal, and communicate this information to the cell death machinery, and to identify components in the pathway that can be manipulated to influence cell survival. Two specific aims are outlined: (1) Define the mechanism(s) by which ER stress activates Caspase-2, and (2) Determine the role of this SH2/SH3-containing adaptor protein in ER stress signaling. These studies will define the mechanisms that control apoptosis downstream of ER stress-a pathway that may represent a key therapeutic target in cancer cells.