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: 7054772
• 负责人: JOHN A.A. LADIAS
• 金额: $ 29.05万
• 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/7054772
• 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对淀粉样蛋白前体蛋白（APP）信号的响应人类基因表达的转录调节的分子机制。 APP是一种跨膜蛋白，是β-淀粉样蛋白肽（ABETA）的前体，β-淀粉样蛋白（ABETA）是淀粉样蛋白斑块的主要组成部分，淀粉样蛋白斑块是阿尔茨海默氏病的特征。该肽是由β-分泌酶通过蛋白水解生成的。 蛋白水解后，APP（APP-C）释放的47个残留细胞质结构域与蛋白质FE65和TIP60相互作用，形成了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氏病发病机理中具有至关重要的重要性，但启动TIP60/FE65/APP-C复合物的序列特异性DNA结合转录因子以及人类基因启动子的启动子以及该复合物通过这种复合物进行转录调节的分子机制的启动。 TIP60具有具有独特底物特异性的组蛋白乙酰转移酶（HAT）活性，并与I类核受体相互作用，这支持了这些转录因子可以介导细胞核中APP-C信号传导的假设。该建议将检验这一假设，并将重点介绍基因调控中APP-C信号传导的机理分析。具体目的是： 1）使用蛋白质 - 蛋白质相互作用测定法与位置定向的诱变和瞬态转染实验结合使用蛋白质 - 蛋白质相互作用测定法，通过与雄激素和雌激素受体的关联来解剖TIP60/FE65/APP-C复合物的基因调节的分子机制。 2）阐明TIP60 HAT底物特异性的结构基础以及TIP60与雄激素和雌激素受体相互作用的结构决定因素。 3）使用X射线晶体学通过App-C结合确定App-C结合在TIP60/FE65复合物上引起的三维构象变化。 这些研究将提供TIP60，FE65和APP-C蛋白的高分辨率三维结构，并将为响应APP信号传导的调节提供机械见解。这些信息可用于制定治疗阿尔茨海默氏病的新策略。