APP MEDIATED SIGNALING EVENTS IN P53 INHIBITION

P53 抑制中应用程序介导的信号事件

• 批准号: 2855089
• 负责人: XIAO XU
• 金额: $ 8.9万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2855089
• 关键词: Alzheimer's disease; aging; amyloid proteins; apoptosis; biological signal transduction; cell age; cell line; chimeric proteins; enzyme linked immunosorbent assay; gel mobility shift assay; neural degeneration; neuropathology; p53 gene /protein; phosphorylation; posttranslational modifications; protein binding; protein structure function; receptor expression; site directed mutagenesis

Alzheimer's disease (AD) is the most common cause of progressive cognitive deficits in elderly people. This as yet incurable disease threatens the health of an increasing number of the elderly in the United States. The financial costs of caring for these patients are enormous. Evidence suggests that APP plays an important role in AD pathogenesis. However, basic functions of APP in the CNS remain largely obscure. Our long term goals are to characterize the functions of APP in the CNS and to determine if age-related dysfunction of APP contributes to neurodegeneration in AD. In our preliminary studies, we found that wild-type, but not familial AD (FAD)-linked APP, protected against apoptosis in an APP-deficient neuroblastoma cell line (B103). APP's antiapoptotic function correlated with its ability to inhibit a key pro-apoptotic molecule, tumor suppress factor p53. In the proposed studies, we will dissect mechanisms underlying APP-mediated inhibition of p53 and apoptosis. First, we will determine if APP decreases p53 activity by inhibiting p53 C-terminal phosphorylation, which regulates p53 activation. Neuronal cell cultures will be challenged with different apoptosis inducers, and the effect of APP on p53 C-terminal phosphorylation will be measured by western blot analysis with antibodies specific to the phosphorylated C-terminus of p53. The APP's ability to inhibit C-terminal phosphorylation and activation of p53 will be analyzed in B103 cells expressing wild-type, FAD-mutant, or frameshift-mutant APP (a truncated APP found in sporadic AD). Next, we will determine the APP-mediated signaling events responsible for p53 inhibition. APP may function as a cell-surface receptor relaying outside signals into cells, which may be responsible for p53 inhibition. To test this hypothesis, we will generate a chimeric APP receptor and determine if chemically-induced dimerizaiton of the chimeric APP receptor will inhibit p53 activation and apoptosis in neuronal cells. If so, we will then determine if the intracellular region of the APP receptor and recently characterized proteins that interact with APP, such as Fe65 and X11, are required for APP's p53 inhibitory function. We will also determine if FAD-linked mutations diminish APP's receptor- mediated function in neuronal cultures. Dissecting the molecular mechanisms underlying APP's regulation of neuronal cell death and survival will greatly expand our understanding of the role of this AD genetic marker in the development of AD.

阿尔茨海默病(AD)是最常见的进行性疾病 老年人的认知缺陷。这还是一种不治之症 威胁着越来越多的老年人的健康 美国。照顾这些病人的经济成本是 巨大的。有证据表明，APP在AD中发挥着重要作用 发病机制。然而，APP在CNS的基本功能仍然很大程度上保持着 默默无闻。我们的长期目标是确定APP的功能 并确定APP是否存在年龄相关性功能障碍 导致阿尔茨海默病的神经退化。在我们的初步研究中，我们 发现野生型，但不是家族性AD(FAD)链接的应用程序，受保护 抗APP缺陷型神经母细胞瘤细胞系(B103)的细胞凋亡。 APP的抗细胞凋亡功能与其抑制一种 关键的促凋亡分子，肿瘤抑制因子P53。在建议的 研究，我们将剖析APP介导的抑制机制 P53与细胞凋亡的关系。首先，我们将确定APP是否会降低P53 通过抑制P53 C末端的磷酸化来调节活性 P53的激活。神经元细胞培养将面临不同的挑战 细胞凋亡诱导物及APP对P53 C末端的影响 磷酸化将通过蛋白质印迹分析来测量 针对P53磷酸化C末端的抗体。这款应用的 抑制P53 C末端磷酸化和激活的能力将 在B103细胞中表达野生型、FAD突变体或 FrameShift-突变应用程序(在零星AD中发现的一种截断的应用程序)。接下来，我们 将确定APP介导的信号事件对P53负责 抑制力。APP可能作为细胞表面受体中继器发挥作用 外界信号进入细胞，这可能是P53抑制的原因。 为了验证这一假设，我们将产生一个嵌合的APP受体，并 确定化学诱导的嵌合APP是否二聚化 受体会抑制神经细胞中P53的激活和凋亡。 如果是这样的话，我们将确定应用程序的细胞内区域 受体和最近鉴定的与APP相互作用的蛋白质， 如FE65和X11是APP的P53抑制功能所必需的。 我们还将确定FAD相关突变是否会削弱APP的受体- 神经元培养中的中介功能。解剖分子 APP调控神经细胞死亡的机制及机制 生存将极大地扩展我们对AD的作用的理解 阿尔茨海默病发生发展中的遗传标记。