FOLLISTATIN--MOLECULAR STRUCTURE/FUNCTION

卵泡抑素--分子结构/功能

• 批准号: 6498126
• 负责人: HENRY T KEUTMANN
• 金额: $ 28.38万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498126
• 关键词: affinity chromatography; aminoacid analyzer; binding sites; crosslink; disulfide bond; follistatin; gel filtration chromatography; high performance liquid chromatography; inhibin; neutralizing antibody; physical model; protein sequence; protein structure function; site directed mutagenesis; structural biology; synthetic peptide

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.

虽然卵泡抑素最初是从卵泡液中分离出来的