MOLECULAR STUDIES OF MONOAMINE OXIDASES

这是研究科学家奖的竞争性续签申请 (RSA)。这项计划的整体目标是进一步研究 单胺氧化酶A和单胺氧化酶的结构、功能及其调控 单胺氧化酶是儿茶酚胺代谢中的一种重要酶。异常 MAO活性的水平已经在许多精神障碍中表现出来。 这些目标将使用人类MAO A、MAO B、cDNA和 本实验室分离的人MAO A和MAO B基因组克隆。 下面介绍这项为期五年的RSA应用程序的具体目标： (I)进一步调查MAO A和MAO B的结构和功能： 负责MAO A和B底物的氨基酸和结构域以及 抑制剂的特异性将通过定点突变进行鉴定。 以及MAO A、MAO B和鲑鱼MAO(鲑鱼)之间构建的嵌合酶 MAO表现出非经典的抑制剂敏感性)。这些变种人会 在酵母中表达。底物的动力学参数(Km)和 将测定抑制剂(KI)。负责以下工作的地区 线粒体靶向将通过使用一系列C- 末端缺失和嵌合突变。动力学参数(Km，Ki) 将对纯化的线粒体进行测定和Western印迹 以证明MAO A或MAO B蛋白是针对线粒体的。 (二)进一步探讨MAO A、MAO B的调控机制 基因表达：MAO A核心启动子上游将有一个新基因 进行了克隆和鉴定。一种新的下调MAO B的抑制子 启动子活性将通过筛选表达文库和 采用亲和柱层析法。特定于细胞的元素(增强剂和 抑制物)和MAO A和B基因表达的转录因子将 通过瞬时表达实验进行鉴定，结合突变， 缺失、凝胶滞留和DNaseI足迹分析。这个 将对MAO A和MAO B的激素刺激机制进行研究。 此外，浓度和DNA对MAO A表达的调节 将研究Sp1在细胞内的结合效率。无论是 老年脑和阿尔茨海默病患者脑内MAO B催化活性升高 患者与改变的启动子序列和/或 将检测转录因子的浓度。这些研究 将提供有关MAO A监管的最基本知识和 B基因表达。新的知识可能最终会导致一部小说 设计调节MAO的特异性MAO抑制剂的方法 在基因水平上的活性，并可能用于精神障碍患者 精神错乱。

This is a competitive renewal application for a Research Scientist Award (RSA). The overall objectives of this project are to further study the structure, function and the regulation of monoamine oxidase (MAO) A and B. MAO is an important enzyme in catecholamine metabolism. Abnormal levels of MAO activity have been shown in a number of mental disorders. These objectives will be accomplished using human MAO A, MAO B, cDNAs and human MAO A and B genomic clones isolated in this laboratory. The specific aims of this five-year RSA application are described below: (I) To further investigate the structure and function of MAO A and B: The amino acids and domains responsible for MAO A and B substrate and inhibitor specificities will be identified by site-directed mutagenesis and chimeric enzymes constructed among MAO A, MAO B and trout MAO (trout MAO exhibits nonclassical inhibitor sensitivities). These mutants will be expressed in yeast. The kinetic parameters for substrates (Km) and inhibitors (Ki) will be determined. The regions responsible for mitochondrial targeting will be identified by using a series of C- terminal deletions and chimeric mutants. The kinetic parameter (Km, Ki) determination and Western blot will be performed on purified mitochondria to demonstrate if the MAO A or B protein is targeted to mitochondria. (II) To further investigate the mechanisms of regulation of MAO A and B gene expression: A new gene upstream of MAO A core promoter will be cloned and characterized. A novel repressor which down regulates MAO B promoter activity will be isolated by screening an expression library and by affinity column chromatography. Cell-specific elements (enhancers and silencers) and transcription factors for MAO A and B gene expression will be identified by transient expression assay, combined with mutations, deletions, gel retardation and DNaseI footprinting assays. The mechanisms of hormone stimulation of MAO A and B will be investigated. Further, the regulation of MAO A expression by the concentration and DNA binding efficiency of Sp1 in cells will be studied. Whether the increased MAO B catalytic activity in aged human brains and Alzheimer's patients is related to altered promoter sequences and/or the concentrations of transcription factors will be examined. These studies will provide most fundamental knowledge on the regulation of MAO A and B gene expression. The new knowledge may ultimately lead to a novel approach for designing specific MAO inhibitors which modulate MAO activity at the gene level, and may be used for patients with mental disorders.