Gene Regulation Mechanisms by the APP/Fe65/TIP60 Complex

APP/Fe65/TIP60 复合物的基因调控机制

• 批准号: 6598733
• 负责人: JOHN A.A. LADIAS
• 金额: $ 29.75万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6598733
• 关键词: X ray crystallography; acyltransferase; amyloid proteins; androgen receptor; biological signal transduction; clinical research; coenzyme A; confocal scanning microscopy; conformation; enzyme mechanism; estrogen receptors; gel mobility shift assay; gene expression; genetic regulation; immunoprecipitation; molecular genetics; protein protein interaction; protein structure; proteolysis; site directed mutagenesis; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the molecular mechanisms of transcriptional regulation of human gene expression in response to Amyloid Precursor Protein (APP) signaling. APP is a transmembrane protein that is the precursor of the beta-amyloid peptide (Abeta), the major component of amyloid plaques that characterize Alzheimer's disease. This peptide is generated from APP through proteolysis by the beta- and gamma-secretases. Following proteolysis, the released 47-residue cytoplasmic domain of APP (APP-C) interacts with the proteins Fe65 and TIP60, forming a nuclear complex that regulates gene expression. However, despite the central importance of the APP-C in human physiology and Alzheimer's disease pathogenesis, the sequence-specific DNA-binding transcription factors that recruit the TIP60/Fe65/APP-C complex onto the promoters of human genes and the molecular mechanisms underlying the transcriptional regulation by this complex remain elusive. TIP60 has histone acetyltransferase (HAT) activity with unique substrate specificity and interacts with class I nuclear receptors, supporting the hypothesis that these transcription factors may mediate APP-C signaling in the nucleus. This proposal will test this hypothesis and will focus on a mechanistic analysis of the APP-C signaling in gene regulation. The Specific Aims are: 1) To dissect the molecular mechanisms of gene regulation by the TIP60/Fe65/APP-C complex through its association with the androgen and estrogen receptors, using protein-protein interaction assays, in combination with site-directed mutagenesis and transient transfection experiments. 2) To elucidate the structural basis of the TIP60 HAT substrate specificity and the structural determinants of the TIP60 interaction with the androgen and estrogen receptors. 3) To determine the three-dimensional conformational changes induced on the TIP60/Fe65 complex by the APP-C binding, using X-ray crystallography. These studies will provide high-resolution three-dimensional structures of the TIP60, Fe65, and APP-C proteins, and will provide mechanistic insights into the regulation of human gene expression in response to APP signaling. This information could be used for the development of novel strategies in the treatment of Alzheimer's disease.

描述（由申请人提供）：我们的长期目标是阐明响应淀粉样前体蛋白（APP）信号传导的人类基因表达转录调控的分子机制。APP是一种跨膜蛋白，是β-淀粉样肽（Abeta）的前体，β-淀粉样肽是阿尔茨海默病特征性淀粉样斑块的主要成分。该肽由APP通过β-和γ-分泌酶的蛋白水解产生。 蛋白水解后，释放的47个残基的APP胞质结构域（APP-C）与蛋白质Fe 65和TIP 60相互作用，形成调节基因表达的核复合物。然而，尽管APP-C在人类生理学和阿尔茨海默病发病机制中具有重要意义，但将TIP 60/Fe 65/APP-C复合物募集到人类基因启动子上的序列特异性DNA结合转录因子以及该复合物转录调控的分子机制仍然难以捉摸。 TIP 60具有组蛋白乙酰转移酶（HAT）活性，具有独特的底物特异性，并与I类核受体相互作用，支持这些转录因子可能介导细胞核中的APP-C信号转导的假设。该提案将测试这一假设，并将集中在APP-C信号在基因调控的机制分析。具体目标是： 1)利用蛋白质-蛋白质相互作用分析，结合定点诱变和瞬时转染实验，剖析TIP 60/Fe 65/APP-C复合物通过与雄激素和雌激素受体结合进行基因调控的分子机制。 2)阐明TIP 60 HAT底物特异性的结构基础以及TIP 60与雄激素和雌激素受体相互作用的结构决定因素。 3)利用X射线晶体学测定APP-C结合引起的TIP 60/Fe 65复合物的三维构象变化。 这些研究将提供TIP 60，Fe 65和APP-C蛋白的高分辨率三维结构，并将提供对APP信号传导的人类基因表达调控的机制见解。这些信息可用于开发治疗阿尔茨海默病的新策略。