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ANABOLIC HORMONES OF DOMINANT NEGATIVE REGULATION

显性负调节的合成代谢激素

基本信息

批准号：
6628557
负责人：
HONG CHEN
金额：
$ 22.72万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-01 至 2006-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6628557
关键词：
Ceboidea biochemical evolution complementary DNA estrogens gene expression genetic promoter element genetically modified animals hormone binding protein hormone regulation /control mechanism laboratory mouse mammary gland molecular cloning osteoblasts protein structure function transcription factor

项目摘要

Primates of the New World (i.e. South and Central America) or platyrrhines are resistant to the cellular actions of the gonadal steroid hormone 17beta-estradiol. Over the span of the last 50 million years platyrrhines have evolved to accommodate this divergence from the hormone-responsive phenotype characteristic of the only other surviving primate suborder, Catarrhini or Old World primates, which include our own species Homo sapiens. Our central hypothesis states that this hormone-resistant phenotype represents the incorporation of a successful gain-of-function mutation by primordial New World primates that resulted in the constitutive overexpression of a family of dominant- negative-acting hormone response element binding proteins in the heterogeneous ribonucleoprotein (hnRNP) superfamily of nucleic acid binding proteins. Two corollaries to our hypothesis hold that this gain-of-function mutation 1] was designed to interrupt estrogen-directed signal transduction independent of the receptor proteins for the hormone, and 2] has been refined over millions of years to require the cooperative action of an hsp-70-related subfamily of high-capacity, intracellular ligand binding proteins. The purpose of this application is to explore the biochemical basis for this extraordinary and successful experiment of Nature by investigating prototypical members of these two novel families of primate signaling proteins, the estrogen-response element binding (ERE-BP) and the intracellular estrogen binding protein (IEBP). The first specific aim is to dissect the molecular basis for the regulated overexpression of the ERE-BP in platyrrhine cells. This goal will be achieved by the application of state-of-the-art technology in 1] tissue- and cell-specific localization of ERE-BP mRNA and protein, 2] functional analysis of the ERE-BP promoter on both an estrogen-resistant and wild-type background, and 3] targeted overexpression of the ERE-BP in estrogen-responsive tissues (i.e. breast and bone). The second specific aim is to 1] purify IEBP, 2] clone its full- length cDNA, and 3] transiently overexpress that cDNA to determine how IEBP co-legislates estrogen responsiveness in platyrrhines. Because there are Old World primate homologs of these overexpressed New World primate proteins, the ultimate aim of this research program is to employ Nature's as well as our own transgenic experiment and to understand the roles of these dominant-negative-acting proteins in estrogen-modified human health and disease (i.e. osteoporosis, breast cancer).

新世界（即南美洲和中美洲）的灵长类动物或长颈犀牛对性腺类固醇激素17 -雌二醇的细胞作用有抵抗力。在过去的5000万年里，鸭嘴龙已经进化到适应这种与其他唯一幸存的灵长类亚目——卡塔利尼或旧大陆灵长类动物（包括我们自己的物种智人）——的激素反应表型特征的差异。我们的中心假设认为，这种激素抗性表型代表了原始新世界灵长类动物成功的功能获得突变的结合，导致异质核糖核蛋白（hnRNP）核酸结合蛋白超家族中显性负作用激素反应元件结合蛋白家族的组成性过表达。我们假设的两个推论认为，这种功能获得突变[1]被设计为中断独立于激素受体蛋白的雌激素定向信号转导，[2]已经经过数百万年的完善，需要高容量细胞内配体结合蛋白的hsp-70相关亚家族的协同作用。本申请的目的是通过研究这两个灵长类信号蛋白新家族的原型成员，雌激素反应元件结合蛋白（雌激素- bp）和细胞内雌激素结合蛋白（雌激素- bp），探索《自然》杂志这一非凡而成功的实验的生化基础。第一个特定的目的是剖析在platyrrhine细胞中调控的ERE-BP过表达的分子基础。这一目标将通过应用最先进的技术来实现：1]ERE-BP mRNA和蛋白的组织和细胞特异性定位；2]雌激素抗性和野生型背景下ERE-BP启动子的功能分析；3]雌激素应答组织（即乳房和骨骼）中ERE-BP的靶向过表达。第二个具体目标是：1]纯化IEBP， 2]克隆其全长cDNA， 3]瞬时过表达该cDNA，以确定IEBP如何共同立法platyrrhines的雌激素反应。由于这些过度表达的新世界灵长类动物蛋白与旧大陆灵长类动物有同源性，因此本研究项目的最终目的是利用自然和我们自己的转基因实验，了解这些显性负作用蛋白在雌激素修饰的人类健康和疾病（如骨质疏松症、乳腺癌）中的作用。