PROHORMONE PROCESSING MODEL IN APLYSIA

多肽激素和神经肽最初是作为前体合成的 随后被有限的蛋白分解所切割的蛋白质。像差 这一过程可能与某些特发性激素疾病有关 功能减退。尽管已经积累了大量关于 前激素及其裂解的分子特征和作用 多肽产品，关于分子的信息有限 酶的特性、作用和定位 负责他们的处理。一个主要问题是与世隔绝 从相对复杂的组织中提取微量的加工酶。 这项提案中描述的研究利用了一种独特的激素前体 海兔体内与产卵激素相关的加工系统 (ELH)，其中识别出的许多问题 哺乳动物系统中的激素前体处理机制被最小化。Elh 是一种神经肽，可以诱导海兔的产卵行为，并且是 腹壁袋状细胞神经元合成前体蛋白 神经节。海兔还有一个外分泌器官，即心房腺， 它表达与ELH相关的基因，并产生多肽 与ELH同源，注射入体内会诱导产卵行为 一只野兽。由于心房腺产生大量与ELH相关的 前体蛋白很容易获得，它的前激素样加工 机制并不复杂，它提供了一个难得的分离机会 前激素样加工酶。袋状细胞神经元可能利用 处理与心房中的酶相同或相似的酶 腺体，因为形成的前体和多肽产品是同源的。 袋状细胞神经元的处理机制表明 与哺乳动物系统相比，复杂性中等，很可能 涉及打包和路由机制。建议进行研究以 分离与ELH相关的二碱性内切酶 心房腺的前体，确定它的结构，建立它的 细胞内定位，并证明其在颗粒内的活性。这些 研究将使用生化和分子克隆进行 技术、Northern杂交、免疫细胞化学和原位杂交 杂交。将开发单抗探针来识别 裂解颗粒内特定的二碱性部位 免疫细胞化学。该模型的开发将从 并将扩大到包括袋状细胞神经元 与其他表达ELH样基因的神经元一样。

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.