Gene Regulation Mechanisms by the APP/Fe65/TIP60 Complex

描述(申请人提供)：我们的长期目标是阐明淀粉样前体蛋白(APP)信号对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-淀粉样多肽(Abeta)的前体，Abeta是阿尔茨海默病淀粉样斑块的主要成分。这种多肽是由APP通过β-和γ-分泌酶的蛋白分解而产生的。 蛋白降解后，APP释放的47个残基的胞质结构域(APP-C)与蛋白质Fe65和Tip60相互作用，形成调控基因表达的核复合体。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核心作用，但将Tip60/Fe65/APP-C复合体招募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体转录调控的分子机制仍不清楚。 Tip60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核内APP-C信号转导的假说。这项提案将检验这一假说，并将重点放在APP-C信号在基因调控中的机制分析上。具体目标是： 1)通过蛋白质-蛋白质相互作用实验，结合定点突变和瞬时转染实验，分析Tip60/Fe65/APP-C复合体与雄激素和雌激素受体结合的基因调控的分子机制。 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)用X射线结晶学方法研究APP-C结合引起的Tip60/Fe65复合体的三维构象变化。 这些研究将提供Tip60、Fe65和APP-C蛋白的高分辨率三维结构，并将提供对APP信号反应中人类基因表达调控的机械性见解。这些信息可以用于开发治疗阿尔茨海默病的新策略。

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.