FOLLISTATIN--MOLECULAR STRUCTURE/FUNCTION

虽然卵泡抑素最初是从卵泡液中分离出来的， 垂体FSH分泌的调节剂，它已迅速获得认可 作为许多组织中细胞发育的重要局部介质 通过其强大的结合激活素的能力。 除了生殖 卵泡抑素影响软骨细胞和破骨细胞的发育 在软骨内骨形成期间，并且参与造血， 胰岛细胞功能和神经发育。 288个残基分子 包括三个独特的10-半胱氨酸结构域，前面是63个残基， N-末端区段，其对于激活素结合似乎是重要的。 值得注意的是，卵泡抑素结构域已经在许多肿瘤细胞中发现。 细胞外基质蛋白，其中一些也结合生长因子， 它们对细胞与细胞的相互作用产生不同的影响， 分化 关于其功能意义知之甚少 卵泡抑素结构域，也不是激活素结合的全部范围， 区域及其对三维结构的依赖性 分子。 该项目将使用蛋白质化学，免疫学和 分子生物学方法来定义关键结构元素 潜在的卵泡抑素作用。 具体目标1将决定 卵泡抑素二硫键排列的分子结构 使用肽图谱和序列分析来确定连接。 结果将 揭示结构域是否代表“自主”折叠单位或 彼此之间， 在目标2下，我们将绘制激活素结合 使用合成肽，位点- 特异性抗体和反应性氨基酸的化学修饰 侧链 将通过激活素竞争试验来测试效果 体外前列腺中激活素活性的结合和抑制 癌细胞系 重组全长卵泡抑素和N-末端 结构域将在Aim 3下表达，并且激活素结合 通过定点诱变精制的要求。 目标4的工作 将建立卵泡抑素结构域的功能作用， 缺失突变体、单个结构域的表达和诱变 在这些领域内的网站。 这将澄清是否 促滤泡素抑制素结构域主要是结构元件或直接 参与激活素结合的N-末端结构域。 一 卵泡抑素分子的功能模型应该会出现， 加强今后在了解其结构基础方面的进展， 在许多系统中，它被发现。

Although follistatin was initially isolated from follicular fluid as a regulator of pituitary FSH secretion, it has rapidly gained recognition as an important local mediator of cell development in many tissues through its potent ability to bind activin. Besides reproductive function, follistatin influences chondrocyte and osteoclast development during endochondral bone formation, and is involved in hematopoeisis, islet cell function, and neural development. The 288-residue molecule includes three distinctive 10-cysteine domains, preceded by a 63-residue N-terminal segment that appears important for activin binding. Significantly, follistatin domains have been found in a number of extracellular matrix proteins, some of which also bind growth factors, that exert diverse effects on cell-cell interactions and differentiation. Little is known regarding the functional significance of the follistatin domains, nor the full extent of the activin-binding region and its dependence on the three-dimensional structure of the molecule. This project will use protein-chemical, immunological and molecular-biological methods to define the critical structural elements underlying follistatin action. Specific Aim 1 will determine the molecular architecture of follistatin by alignment of the disulfide linkages using peptide mapping and sequence analysis. The results will reveal whether the domains represent "autonomous" folding units or are crosslinked to one another. Under Aim 2 we will map the activin-binding determinants in the N-terminal domain using synthetic peptides, site- specific antibodies, and chemical modification of reactive amino-acid side-chains. Effects will be tested by competition assays for activin binding and inhibition of activin activity in an in vitro prostate cancer cell line. Recombinant full-length follistatin and N-terminal domain will be expressed under Aim 3 and the activin binding requirements refined by site-directed mutagenesis. The work of Aim 4 will establish the functional role of the follistatin domains through deletion mutants, expression of an individual domain, and mutagenesis at sites within these domains. This will clarify whether the follistatin domains are primarily structural elements or direct participants with the N-terminal domain in activin binding. A functional model of the follistatin molecule should emerge that will enhance future progress in understanding the structural basis for its actions in the many systems in which it is found.