Prohormone processing: NPY/Catestatin Peptide Production

激素原加工：NPY/儿茶素肽生产

• 批准号: 7122643
• 负责人: Vivian Y. H Hook
• 金额: $ 28.48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122643
• 关键词: antisense nucleic acid; blood pressure; cardiovascular function; chromaffin cells; cysteine endopeptidases; enzyme activity; gene expression; high performance liquid chromatography; mass spectrometry; molecular cloning; neuropeptide Y; peptide hormone biosynthesis; prohormone convertase; protease inhibitor; protein localization; protein structure function; sympathetic nervous system; tissue /cell culture; western blottings

Neuropeptide Y (NPY) and catestatin peptides are secreted from adrenomedullary chromaffin cells and sympathetic nerves for the regulation of blood pressure. NPY acts as a direct vasoconstrictor, and catestatin functions as an autocrine regulator to inhibit nicotine-stimulated catecholamine release. Elevated NPY and reduced catestatin in essential hypertension implicate their participation as neuroeffectors in regulating blood pressure. Importantly, knowledge of the major proteolytic enzymes (s) responsible for converting their respective prohormone precursors into active NPY and catestatin is crucial for understanding regulatory mechanisms that control blood pressure. The prohormone precursors of NPY and catestatin, pro-NPY and chromogranin A (CgA), respectively, undergo proteolytic processing within secretory vesicles of adrenal medulla, known as chromaffin granules. We have identified secretory vesicle cathepsin L, previously known as ?prohormone thiol protease? (PTP), as a key processing enzyme for pro-NPY and CgA. Moreover, this project discovered a novel endogenous serpin, endopin 2 that inhibits secretory vesicle cathepsin L. In addition, the subtilisin-like PC1 and PC2 proteases in secretory vesicles may also participate in pro-NPY and CgA processing. Based on these new findings, the goal of Project 3 will be to assess the roles of secretory vesicle cathepsin L and endopin 2, compared to PC1 and PC2, in the production of NPY and catestatin neuropeptides that regulate blood pressure. This project will test the hypothesis that secretory vesicle cathepsin L may be a major processing enzyme for NPY and catestatin, compared to PC1 and PC2 enzymes. Our new results support the emerging biological role of cathepsin L function in secretory vesicles for proteolysis of pro-NPY and CgA. Moreover, our recent studies of cathepsin L knockout mice suggest participation of this protease in NPY production in adrenals. These results lead to the next phase of this study that will (1) evaluate in vitro and cellular processing of pro-NPY and CgA by secretory vesicle cathepsin L, compared to PC1 and PC2, (2) assess the cellular and tissue distribution of cathepsin L and PC enzymes in secretory vesicles that contain NPY and catestatin, (3) conduct cellular antisense and gene knockout studies to examine the relative roles of cathepsin L and PC enzymes for NPY and catestatin production, and (4) evaluate endopin 2 as an endogenous serpin inhibitor of cathepsin L for neuropeptide production. Results will demonstrate the relative roles for cathepsin L and endopin 2, compared to PC1 and PC2, in the biosynthesis of active NPY and catestatin peptide regulators. Project 3 complements the program project theme of understanding the regulation of sympathetic neuroeffectors that participate in blood pressure regulation.

神经肽Y(NPY)和儿茶素多肽是肾上腺髓质嗜铬细胞和交感神经分泌的调节血压的多肽。NPY起到直接的血管收缩作用，儿茶素起到自分泌调节的作用，从而抑制尼古丁刺激的儿茶酚胺的释放。高血压患者NPY水平升高和儿茶素水平降低，提示它们参与了血压调节的神经效应。重要的是，了解负责将各自的前激素前体转化为活性神经肽Y和儿茶素的主要蛋白水解酶(S)对于理解控制血压的调节机制至关重要。NPY和儿茶素的前体分别是原NPY和嗜铬粒蛋白A(Chromoranin A，CGA)，它们分别在肾上腺髓质的分泌小泡内进行蛋白质分解处理，称为嗜铬颗粒。我们已经鉴定出分泌性囊泡组织蛋白L，以前被称为？激素原硫醇蛋白酶？(PTP)，作为前NPY和CGA的关键加工酶。此外，本项目还发现了一种新的内源性丝氨酸，内毒素2，它能抑制分泌性囊泡组织蛋白L。此外，类枯草杆菌菌素 分泌囊泡中的PC1和PC2蛋白水解酶也可能参与前NPY和CGA的加工。基于这些新发现，项目3的目标将是评估分泌囊泡组织蛋白L和内毒素2与PC1和PC2在产生调节血压的神经肽Y和儿茶素神经肽中的作用。该项目将检验一种假设，即分泌囊泡组织蛋白L可能是神经肽Y和儿茶素的主要加工酶，而不是PC1和PC2酶。我们的新结果支持了组织蛋白酶L在分泌囊泡中对前体NPY和CGA的蛋白分解所起的生物学作用。此外，我们最近对组织蛋白酶L基因敲除小鼠的研究表明，这种蛋白酶参与了肾上腺神经肽Y的产生。这些结果将导致下一阶段的研究，将(1)评估组织蛋白酶L对神经肽Y和CgA原的体外和细胞处理，并与PC1和PC2进行比较；(2)评估组织蛋白酶L和PC酶在含有NPY和儿茶素的分泌囊泡中的细胞和组织分布；(3)进行细胞反义和基因敲除研究，以研究组织蛋白酶L和PC酶在NPY和儿茶素产生中的相对作用；以及(4)评估内毒素2作为组织蛋白酶L的内源性丝氨酸抑制物，用于神经肽的产生。结果将证明组织蛋白酶L和内毒素2在活性神经肽Y和儿茶素多肽的生物合成中相对于PC1和PC2的相对作用。项目3补充了项目主题，即了解参与血压调节的交感神经效应器的调节。