CELL BIOLOGY OF OVARIAN APOLIPOPROTEIN E

卵巢载脂蛋白 E 的细胞生物学

• 批准号: 2200252
• 负责人: CHERYL A DYER
• 金额: $ 8.26万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2200252
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; affinity chromatography; androgen inhibitor; apolipoproteins; autoradiography; binding proteins; cell differentiation; chemical binding; cyclic AMP; graafian follicles; granulosa cell; human tissue; immunocytochemistry; in situ hybridization; iodine; laboratory rabbit; laboratory rat; ligands; low density lipoprotein; messenger RNA; ovary; peptide analog; plasma; protein biosynthesis; protein structure function; radionuclides; radiotracer; steroid hormone biosynthesis; tissue /cell culture

Apoprotein (apo) E is a well-characterized protein whose acknowledged function is to mediate the transport and uptake of cholesterol-rich plasma lipoproteins. Incongruous with this role in cholesterol homeostasis is the repeated observation that apo E is also synthesized in multiple extrahepatic sites including ovarian granulosa cells. While investigating the lipoprotein requirements of ovary androgen producing cells, I discovered that apo E is a specific inhibitor of androgen synthesis. In the presence of apo E, cultured LH-stimulated theca/interstitial (T/I) cells make progesterone, but cannot convert it to androgen. Furthermore, I have recently identified a synthetic peptide analogue of apo E that is capable of mimicking intact apo E. Granulosa cells and theca cells are dependent on one another to coordinate the production of estrogen that is critical in controlling follicular development. To test the hypothesis that granulosa cell apo E is an intraovarian regulator of T/I cell androgen production, five Specific Aims will be accomplished. The first is to demonstrate that granulosa cell-derived apo E inhibits T/I cell androgen production. Apo E will be isolated from granulosa cell culture supernatants and tested for biologic activity. The second Specific Aim is to characterize the changes in ovarian apo E content, apo E synthesis, and apo E binding during follicular development. Using immunohistochemical and autoradiographic techniques, apo E and its binding sites will be identified in follicles at different stages of development. In addition, cells expressing apo E mRNA will be identified by In situ hybridization. The third Specific Aim is to characterize the interaction of apo E with cultured T/I cells and to identify the molecular nature of the cellular binding sites. Using both granulosa cell-derived apo E and a synthetic apo E peptide analogue, binding to T/I cells will be characterized. The fourth Specific Aim is to define the structural features of apo E that are responsible for its binding to and biologic effect on T/I cells. Chemical modifications and amino acid substitutions will be made in the apo E synthetic peptide to define the structural requirements for both binding and function. The fifth Specific Aim is to identify the site of the inhibitory activity of apo E pre- and post-cAMP mediation of theca/interstitial cell differentiation. Based on the information obtained from this investigation, it will be possible to assess the physiologic role of granulosa cell apo E in ovarian function.

丙蛋白（apo）E是一种良好的蛋白质 公认的功能是调解运输和吸收 富含胆固醇的血浆脂蛋白。 与这个角色不协调 胆固醇稳态是反复观察到apo e也是 在包括卵巢颗粒在内的多个肝外部位合成 细胞。 在研究卵巢的脂蛋白需求时 雄激素产生细胞，我发现apo e是一种特定的抑制剂 雄激素合成。 在apo e的存在下，培养的LH刺激 theca/间质（T/I）细胞会产生孕激素，但无法转换 到雄激素。 此外，我最近确定了合成肽 能够模仿完整apo E.颗粒的apo e的类似物 细胞和theca细胞彼此依赖以协调 产生雌激素，这对于控制卵泡至关重要 发展。 测试颗粒细胞apo e的假设是 T/I细胞雄激素产生的载体内调节剂，五个特异性 目标将实现。 首先是证明颗粒 细胞来源的Apo E抑制T/I细胞雄激素的产生。 apo e将会 从颗粒细胞培养上清液中分离并测试生物学 活动。 第二个具体目的是表征变化 卵巢apo e含量，apo e合成和apo e在期间结合 卵泡发展。 使用免疫组织化学和放射自显影 技术，apo e及其结合位点将在卵泡中识别 在不同的发展阶段。 另外，表达apo e的细胞 mRNA将通过原位杂交识别。 第三个特定目标 是为了表征Apo e与培养的T/I细胞的相互作用以及 确定细胞结合位点的分子性质。 使用 颗粒细胞衍生的apo e和合成apo e肽模拟， 将表征与T/I细胞的结合。 第四个特定目标是 定义apo e的结构特征 与T/I细胞的结合和生物学作用。 化学修饰和 将在Apo e合成肽中进行氨基酸取代 定义结合和功能的结构要求。 这 第五个具体目的是确定 theca/间质细胞的训练前和训练后调解 分化。 根据从此获得的信息 调查，可以评估 卵巢功能中的颗粒状细胞apo e。