PROHORMONE PROCESSING MODEL IN APLYSIA

海兔的激素原加工模型

• 批准号: 3416059
• 负责人: ALEXANDER none KUROSKY
• 金额: $ 15.43万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3416059
• 关键词: Aplysia; endopeptidases; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; high performance liquid chromatography; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; invertebrate hormone; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; neuropeptides; nucleic acid probes; peptide hormone; protein purification; protein sequence

Peptide hormones and neuropeptides are initially synthesized as precursor proteins that are subsequently cleaved by limited proteolysis. Aberrations of this process may be responsible for some diseases of idiopathic hormone hypofunction. Although considerable information has accumulated regarding the molecular characterization and action of prohormones and their cleaved peptide products, there is limited information about the molecular characteristics, action, and localization of the enzymes which are responsible for their processing. A major problem has been the isolation of minute amounts of processing enzymes from relatively complex tissues. Research described in this proposal takes advantage of a unique prohormone processing system in Aplysia californica relating to egg-laying hormone (ELH), in which many of the problems identified in characterizing prohormone processing mechanisms in mammalian systems are minimized. ELH is a neuropeptide which elicits egg-laying behavior in Aplysia and is synthesized as a precursor protein by bag cell neurons of the abdominal ganglion. Aplysia also possesses an exocrine organ, the atrial gland, which expresses genes that are related to ELH, and produces peptides homologous to ELH which will induce egg-laying behavior when injected into an animal. Since the atrial gland produces large quantities of ELH-related precursor protein is readily accessible, and its prohormone-like processing mechanism is not complex, it provides a rare opportunity to isolate prohormone-like processing enzymes. The bag cell neurons probably utilize processing enzymes that are identical or similar to those in the atrial gland, since the precursors and peptide products formed are homologous. Processing mechanisms in the bag cell neurons indicate to be of intermediate complexity when compared to mammalian systems, and are likely to involve packaging and routing mechanisms. Research is proposed to isolate the dibasic endoprotease involved in processing ELH-related precursors in the atrial gland, determine its structure, establish its intracellular location, and demonstrate its intragranular activity. These studies will be carried out using biochemical and molecular cloning techniques, Northern hybridization, immunocytochemistry, and in situ hybridization. Monoclonal antibody probes will be developed to identify the cleavage of specific dibasic sites within granules by immunocytochemistry. Development of the model will initiate with the atrial gland and will be expanded to include the bag cell neurons, as well as other neurons expressing ELH-like genes.

肽激素和神经肽最初是作为前体合成的 随后通过有限的蛋白质水解被切割的蛋白质。 像差 这一过程可能与某些特发性激素性疾病有关 机能减退 尽管已经积累了大量关于 激素原的分子特征和作用及其切割 肽产品，有有限的信息，分子 酶的特性、作用和定位， 负责其处理。 一个主要的问题是与世隔绝 从相对复杂的组织中提取的微量加工酶。 这项提案中描述的研究利用了一种独特的激素原， 与产卵激素有关的加州滨藜加工系统 (ELH)，其中许多问题确定在表征 哺乳动物系统中的激素原加工机制被最小化。 ELH 是一种神经肽，它能刺激果蝇的产卵行为， 作为前体蛋白由腹部的袋细胞神经元合成 神经节 失智症还具有一个外分泌器官，心房腺， 它表达与ELH相关的基因， 与ELH同源，当注射到 一只动物 由于心房腺产生大量ELH相关的 前体蛋白很容易获得，其类似蛋白酶的加工 机制并不复杂，它提供了一个难得的机会， 类似脯氨酸的加工酶。 袋细胞神经元可能利用 与心房中的酶相同或相似的加工酶 腺，因为前体和肽产物形成同源。 袋细胞神经元的加工机制表明， 与哺乳动物系统相比， 涉及打包和路由机制。 研究建议， 分离参与加工ELH相关的二元内切蛋白酶 前体在心房腺，决定其结构，建立其 细胞内定位，并证明其颗粒内活性。 这些 将利用生物化学和分子克隆技术进行研究 技术、北方杂交、免疫细胞化学和原位 杂交方法 将开发单克隆抗体探针， 颗粒内特定二元位点的裂解， 免疫细胞化学 模型的开发将从 心房腺，并将扩大到包括袋细胞神经元，以及 与其他表达ELH样基因的神经元一样。