MECHANISM AND REGULATION OF FOLLISTATIN PROCESSING

卵泡抑素（FS）是一种单链糖蛋白，其已被证明不可逆地结合并中和活化素，活化素是TGF-β超家族中的多效性生长因子。 虽然从一个单一的基因转录，选择性剪接和翻译后蛋白水解修饰产生3 FS核心蛋白不同的C-末端，组织分布，并在其生化和生理特性。由于激活素是卵泡发生、骨形成、组织命运决定和其他发育和生理过程的关键调节剂，并且FS表达与激活素的表达在很大程度上重叠，因此FS实际上在控制激活素的生物学作用中充当关键的“开/关开关”。此外，激活素是卵泡发育中颗粒细胞增殖和分化的关键调节因子。由于FS的产生在整个卵泡发育过程中增加，颗粒细胞分泌激活素和FS，很明显，这两种分子之间的相互作用影响了颗粒细胞和卵泡在正常月经周期中的成熟。此外，我们的初步研究表明，不同的FS形式可能具有不同的激活素调节功能。因此，该提案的广泛目标是定义FS 315的转录后蛋白水解加工的调节机制和细胞后果，该过程最终决定FS在体内和发育卵泡中的功能和分布。在第一个具体目标中，我们建议使用我们已经证明具有这种重要蛋白水解活性的细胞系分离和纯化FS 315加工蛋白酶。一旦克隆，将确定表达和活性的激素调节。第二个特定目标重点关注这种蛋白酶的细胞内位置以及细胞内外加工和未加工FS的分布。在第三个具体目标中，我们将描述未加工和加工FS之间的功能差异，检查FS和激活素的细胞外和潜在的细胞内活性。这些研究将导致更好地了解不同形式的FS是如何在不同组织中产生的，以及它们的生物活性，这是阐明FS在人体中的生理学的必要先决条件。

Follistatin (FS) is a single chain glycoprotein that has been shown to irreversibly bind and neutralize activin, a pleotropic growth factor in the TGF-beta superfamily. Although transcribed from a single gene, alternative splicing and post-translational proteolytic modifications produce 3 FS core proteins differing in their c-terminus, tissue distribution, and in their biochemical and physiological properties. Since activin is a critical regulator of ovarian folliculogenesis, bone formation, tissue fate determination, and other developmental and physiological processes, and FS expression largely overlaps that of activin, FS actually functions as a critical "on/off switch" in governing the biological actions of activin. In addition, activin is a critical regulator of granulosa cell proliferation and differentiation within developing follicles. Since FS production increases throughout follicular development and granulosa ells secrete both activin and FS, it is clear that the interplay between these two molecules influences how granulosa cells and follicles mature during the normal menstrual cycle. Moreover, our preliminary studies indicate that the different FS forms may have different activin-regulating functions. Thus, the broad goal of this proposal is to define the regulatory mechanisms and cellular consequences of post-transcriptional proteolytic processing of FS315, a process which ultimately determines the function and distribution of FS within the body and developing follicle. In the first Specific Aim, we propose to isolate and purify the FS315 processing protease using a cell line that we have demonstrated has this important proteolytic activity. Once cloned, hormonal regulation of expression and activity will be determined. The second Specific Aim focuses on the intracellular location of this protease and the distribution of processed and unprocessed FS inside and outside the cell. In the third Specific Aim we will characterize the functional differences between unprocessed and processed FS, examining both extracellular and potential intracellular activities of FS and activin. The studies will result in a better understanding of how different forms of FS are produced in different tissues as well as their biological activities, a necessary prerequisite to elucidate the physiology of FS in the human.