ANABOLIC HORMONES OF DOMINANT NEGATIVE REGULATION

新大陆（即南美洲和中美洲）的灵长类动物或阔鼻类动物对性腺类固醇激素17 β-雌二醇的细胞作用具有抗性。 在过去的5000万年里，阔鼻类已经进化到适应这种与其他唯一幸存的灵长类亚目--卡他鼻类或旧大陆灵长类--的对气味反应的表型特征的差异，其中包括我们自己的物种智人。 我们的中心假设指出，这种耐药表型代表了原始新世界灵长类动物成功的功能获得性突变的掺入，该突变导致核酸结合蛋白的异质核糖核蛋白（hnRNP）超家族中的显性负作用激素反应元件结合蛋白家族的组成性过表达。 我们的假设的两个推论认为，这种功能获得性突变1]被设计为中断雌激素介导的信号转导，而不依赖于激素的受体蛋白，2]已经经过数百万年的改进，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新的灵长类动物信号蛋白家族的原型成员，雌激素反应元件结合（ERE-BP）和细胞内雌激素结合蛋白（IEBP），探索自然界这一非凡而成功的实验的生化基础。 第一个具体的目的是解剖的调控过表达的ERE-BP在阔鼻细胞的分子基础。 这一目标将通过在以下方面应用最先进的技术来实现：1] ERE-BP mRNA和蛋白质的组织和细胞特异性定位，2] ERE-BP启动子在雌激素抗性和野生型背景下的功能分析，以及3] ERE-BP在雌激素应答组织（即乳腺和骨）中的靶向过表达。第二个具体目的是1]纯化IEBP，2]克隆其全长cDNA，和3]瞬时过表达该cDNA以确定IEBP如何在阔鼻鱼中共同调节雌激素反应性。 由于这些过度表达的新世界灵长类动物蛋白存在旧世界灵长类动物同源物，因此本研究计划的最终目的是采用自然界以及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（即骨质疏松症，乳腺癌）中的作用。

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).