ANABOLIC HORMONES OF DOMINANT NEGATIVE REGULATION

显性负调节的合成代谢激素

• 批准号: 6350733
• 负责人: HONG CHEN
• 金额: $ 23.01万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350733
• 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

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).

新大陆(即南美洲和中美洲)的灵长类动物或鸭嘴兽对性腺类固醇激素17β-雌二醇的细胞活动具有抵抗力。在过去的5000万年里，鸭嘴兽的进化适应了与唯一幸存的其他灵长类亚目--卡特雷尼亚目或东半球灵长类动物--的激素反应表型的这种差异，其中包括我们自己的物种智人。我们的中心假设是，这种激素抗性表型代表着原始新大陆灵长类动物成功地加入了一个功能获得突变，导致了一系列显性-负向作用的激素反应元件结合蛋白在异质性核糖核蛋白(HnRNP)超家族核酸结合蛋白中的结构性过表达。我们假设的两个推论认为，这种功能获得突变1]被设计来中断雌激素导向的信号转导，而不依赖于激素的受体蛋白，2]经过数百万年的改进，需要HSP-70相关的高容量细胞内配体结合蛋白亚家族的协同作用。这一应用的目的是通过研究这两个新的灵长类信号蛋白家族的原型成员--雌激素反应元件结合蛋白(ERE-BP)和细胞内雌激素结合蛋白(IEBP)，来探索这一非凡而成功的自然实验的生化基础。第一个特定的目的是剖析在白颈鹿细胞中调节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)组织和细胞特定的ERE-BP mRNA和蛋白质定位；2)在雌激素抗性和野生型背景下对ERE-BP启动子的功能分析；以及3)在雌激素反应的组织(即乳房和骨骼)中有针对性地过表达ERE-BP。第二个特异目的是1)纯化IEBP，2]克隆其全长cDNA3]瞬时过表达该cDNA3)以确定IEBP如何共同调节白颈鹿的雌激素反应。由于这些过度表达的新世界灵长类蛋白存在旧世界灵长类同源物，本研究计划的最终目的是利用自然和我们自己的转基因实验，并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(如骨质疏松症和乳腺癌)中的作用。