Signaling Cell Death from the Endoplasmic Reticulum

内质网发出细胞死亡信号

• 批准号: 7661670
• 负责人: Scott A. Oakes
• 金额: $ 32.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7661670
• 关键词: Adaptor Signaling Protein; Apoptosis; Apoptotic; BAX gene; BID protein; Caspase; Cell Death; Cell Death Signaling Process; Cell Survival; Cells; Cellular Stress; Cessation of life; Cleaved cell; Complex; Coupled; Data; Defect; Endoplasmic Reticulum; Environment; Event; Homeostasis; Hypoxia; In Vitro; Laboratories; Lead; Life; Location; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Membrane; Mitochondria; Molecular; Molecular Chaperones; Neoplasm Metastasis; Nutrient; Outer Mitochondrial Membrane; Output; Oxygen; Pathway interactions; Process; Protein Family; Proteins; Proto-Oncogenes; Role; SH3 Domains; Sequence Analysis; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stress; Supporting Cell; Technology; Therapeutic; Translations; Vascular blood supply; accomplished suicide; cancer cell; caspase-2; cytotoxic; deprivation; endoplasmic reticulum stress; in vivo; killings; neoplastic cell; new therapeutic target; novel; pro-caspase-2; programs; protein folding; protein misfolding; public health relevance; response; sensor; src Homology Region 2 Domain; therapeutic target

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. PUBLIC HEALTH RELEVANCE: All cells in our body are genetically programmed to commit suicide through a process called "apoptosis" when exposed to stressful conditions such as low oxygen or scarce blood supply. Defects in this apoptotic pathway allow cancer cells to survive and metastasize to foreign environments where unfavorable conditions would normally trigger death. This projects sets out to define how cellular stress normally leads to apoptosis and what goes wrong with this process in cancer-in the hopes of finding new therapeutic targets through which to kill tumor cells.

描述（由申请人提供）：癌细胞必须避开细胞凋亡检查点才能在不利条件下生存，例如营养缺乏和缺氧，这很快会导致对内质网（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程度达到临界水平时，细胞会参与一组进化上保守的信号转导途径，这些途径统称为未折叠蛋白反应（UPR）。哺乳动物细胞中 UPR 的主要效应器是 ER 驻留跨膜传感器 IRE11、PERK 和 AFT6。这些压力传感器最初扩展 ER 网络，上调伴侣并阻止全局翻译以恢复体内平衡。然而，如果内质网损伤严重，这些内质网驻留应激传感器就会通过人们知之甚少的机制启动细胞凋亡。许多癌症都存在持续且高水平的内质网应激；因此，恶性细胞必须进化出机制来逃避这种应激的正常细胞毒性后果。恢复 UPR 的凋亡输出的努力有望成为杀死癌细胞的治疗策略。过度的内质网应激会触发“内在”凋亡途径，该途径在线粒体外膜上受到促死亡 BCL-2 家族蛋白 BAX 和 BAK 的严格调节。然而，从 ER 应激到线粒体 BAX/BAK 激活的分子链事件仍然知之甚少。我的实验室开发了一种方法，可以从 ER 应激的 Bax-/-Bak-/- 细胞的胞质提取物中纯化线粒体前凋亡活性。利用这项技术，我们确定了聚集在线粒体 BAX/BAK 上的两个主要细胞凋亡信号。一种信号是 BH3-only 蛋白 BID，它被 Caspase-2 切割成更短的促凋亡形式。我们现在试图了解从内质网膜错误折叠蛋白的感知到 Caspase-2（最难表征的哺乳动物半胱天冬酶之一）催化激活的事件。我们最近还从活性提取物中纯化了第二种新成分——含有一个 SH2 结构域和两个 SH3 结构域的接头蛋白，我们的数据表明它是 ER 应激下游的不依赖于 BID 的细胞凋亡信号。我们现在的目标是确定该接头蛋白在内质网应激信号传导中的促凋亡作用。该提案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，并将此信息传达给细胞死亡机制，并识别通路中可操纵以影响细胞存活的成分。概述了两个具体目标：(1) 定义 ER 应激激活 Caspase-2 的机制，以及 (2) 确定这种包含 SH2/SH3 的衔接蛋白在内质网应激信号传导中的作用。这些研究将确定控制内质网应激下游细胞凋亡的机制——这一途径可能代表癌细胞的关键治疗靶点。公共健康相关性：我们体内的所有细胞都经过基因编程，当暴露于低氧或血液供应稀缺等压力条件时，会通过称为“细胞凋亡”的过程自杀。这种凋亡途径的缺陷使癌细胞能够存活并转移到不利条件通常会引发死亡的异质环境。该项目旨在定义细胞应激通常如何导致细胞凋亡，以及癌症中这一过程出了什么问题，希望找到杀死肿瘤细胞的新治疗靶点。