Prohormone processing: NPY/Catestatin Peptide Production

激素原加工：NPY/儿茶素肽生产

• 批准号: 7312480
• 负责人: Vivian Y. H Hook
• 金额: $ 29.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7312480
• 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) 和儿茶素肽由肾上腺髓质嗜铬细胞和交感神经分泌，用于调节血压。 NPY 作为直接血管收缩剂，而儿茶素作为自分泌调节剂，抑制尼古丁刺激的儿茶酚胺释放。原发性高血压中 NPY 升高和儿联蛋白减少表明它们作为神经效应器参与调节血压。重要的是，了解负责将各自激素原前体转化为活性 NPY 和儿联蛋白的主要蛋白水解酶对于理解控制血压的调节机制至关重要。 NPY 和儿茶素的激素前体、NPY 前体和嗜铬粒蛋白 A (CgA) 分别在肾上腺髓质的分泌囊泡（称为嗜铬颗粒）内进行蛋白水解加工。我们已经鉴定出分泌囊泡组织蛋白酶 L，以前称为“激素原硫醇蛋白酶”？ (PTP)，作为 pro-NPY 和 CgA 的关键加工酶。此外，该项目还发现了一种新型内源性丝氨酸蛋白酶抑制剂（endopin 2），可抑制分泌囊泡组织蛋白酶 L。此外，枯草杆菌蛋白酶（subtilisin-like） 分泌囊泡中的 PC1 和 PC2 蛋白酶也可能参与 pro-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酶 L 和内皮素 2 与 PC1 和 PC2 相比，在调节血压的 NPY 和儿茶素神经肽的产生中的作用。该项目将测试以下假设：与 PC1 和 PC2 酶相比，分泌囊泡组织蛋白酶 L 可能是 NPY 和儿茶素的主要加工酶。我们的新结果支持了组织蛋白酶 L 在分泌囊泡中对 NPY 前体和 CgA 进行蛋白水解的新兴生物学作用。此外，我们最近对组织蛋白酶 L 敲除小鼠的研究表明，这种蛋白酶参与了肾上腺 NPY 的产生。这些结果导致本研究的下一阶段将（1）与PC1和PC2相比，评估分泌囊泡组织蛋白酶L对pro-NPY和CgA的体外和细胞加工，（2）评估组织蛋白酶L和PC酶在含有NPY和catestatin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检查组织蛋白酶L的相对作用 和 PC 酶用于 NPY 和儿茶素生产，(4) 评估内皮素 2 作为组织蛋白酶 L 的内源性丝氨酸蛋白酶抑制剂用于神经肽生产。结果将证明组织蛋白酶 L 和内皮素 2 与 PC1 和 PC2 相比，在活性 NPY 和儿茶素肽调节剂的生物合成中的相对作用。项目 3 补充了了解参与血压调节的交感神经效应器的调节的项目主题。