Novel Functions of the Ah Receptor

超出提供的空间。芳香烃受体(AhR)的生物学功能通常归因于其作为配体激活的转录因子的功能。然而，最近的研究表明，AhR可能具有配体非依赖的功能。对具有不同AhR含量的小鼠肝癌细胞的研究表明，AhR含量与神经酰胺诱导的细胞凋亡的敏感性直接相关，但与其他因素无关。最近的研究表明，神经酰胺可以引起含AhR细胞的溶酶体破坏、胞质BID的裂解、细胞色素c的释放和线粒体凋亡途径的激活。神经酰胺的作用可以通过光动力疗法(PDT)靶向破坏溶酶体来复制。由于光动力疗法(PDT)和神经酰胺破坏了AhR缺陷细胞的溶酶体，但不引起BID裂解，我们假设：1)溶酶体的破坏触发了线粒体！2)AhR调控参与BID裂解的过程。在本申请中，我们建议使用BID零和AhR零细胞来确定是否激活了‘线粒体！’。神经酰胺/PDT诱导的细胞凋亡途径分别依赖于1)Bid和2)AhR含量。3)我们将确定我们的AhR缺陷细胞系无法切割BID是否反映了溶酶体BID切割活性的缺乏，或者胞浆蛋白(S)的存在抑制了这一活性。4)我们将尝试通过传统的纯化技术、选择性的药物抑制或蛋白质组学方法来鉴定与BID切割有关的溶酶体活性。5)将突变形式的AhR导入AhR缺陷细胞，将被用于确定区域/功能(例如，DNA和配体结合、异源二聚化、反式激活等)。AhR在调控Bid裂解/神经酰胺诱导的细胞凋亡中起重要作用。最后，将测试一系列“氧化应激源”，以确定氧化剂是否会诱导“线粒体”的激活！细胞凋亡途径是溶酶体破坏和Bid裂解的下游产物。这些研究将提供一种将溶酶体破坏与细胞凋亡联系起来的机制，并表征一种新的AhR功能。溶酶体破坏除引起坏死外，还与许多毒物的作用机制以及与健康相关的领域有关，如神经和肺毒理学、化学和光疗、中风研究和再灌注损伤。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The biological functions of the aryl hydrocarbon receptor (AhR) are normallyattributed to its functioning as a ligand-activated transcription factor. However, recent studies suggest that the AhR may have ligand- independent functions. Studies with murine hepatoma cells engineered to have different AhR contents show a direct correlation between AhR content and susceptibility to apoptosis induced by ceramide, but not other agents. Recent studies have shown that ceramide causes lysosome disruption, cleavage of cytosolic Bid, release of cytochrome c, and activation of the 'mitochondria!'apoptotic pathway in AhR-containing cells. The effects of ceramide could be duplicated by the targeted disruption of lysosomes with photodynamic therapy (PDT). Because PDT and ceramide disrupt the lysosomes of AhR-deficient cells, but do not cause Bid cleavage we hypothesize that 1) lysosome disruption triggers the 'mitochondria!' apoptotic pathway via lysosomal protease cleavage of Bid, and 2) the AhR modulates processes involved in Bid cleavage. In this application we propose to use Bid null and AhR null cells to determine if activation of the 'mitochondria!' apoptotic pathway by ceramide/PDTis dependent upon 1) Bid and 2) AhR content, respectively. 3) We will determine if the inability of our AhR-deficient cell lines to cleave Bid reflects the absence of a lysosomal Bid cleavage activity, or the presence of cytosolic protein(s) that inhibit this activity. 4) We will attempt to identify the lysosomal activity responsible for Bid cleavage by conventional purification techniques, selective pharmacological inhibition, or a proteomic approach. 5) Transfection of mutated forms of the AhR into AhR-deficient cells will be used to determine the regions/functions (e.g., DNA and ligand-binding, heterodimerization, transactivation, etc.) of the AhR important in the modulation of Bid cleavage/ceramide- induced apoptosis. Lastly, 6) a series of 'oxidative stressors' will be tested to determine if oxidant-induced activation of the 'mitochondria!' apoptotic pathway is a downstream consequence of lysosome disruption and Bid cleavage. These studies will provide a mechanism linking lysosome disruption and apoptosis, and characterize a novel AhR function. The concept that lysosome disruption can trigger apoptosis, in addition to necrosis, is relevant to the mechanism of action of many toxicants, and health-related areas such as neuro and pulmonary toxicology, chemo-and phototherapy, stroke research, and reperfusion induced injury. PERFORMANCE SITE ========================================Section End===========================================