Biosynthesis of Enkephalin Opioid Peptides

脑啡肽阿片肽的生物合成

• 批准号: 6723522
• 负责人: Vivian Y. H Hook
• 金额: $ 30.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723522
• 关键词: PC12 cells; aminopeptidase; brain; chemical kinetics; chromaffin cells; cysteine endopeptidases; enkephalins; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme substrate; genetically modified animals; laboratory mouse; laboratory rat; mass spectrometry; molecular cloning; molecular site; neuroendocrine system; prohormone convertase; protein biosynthesis; protein localization; proteolysis; radioimmunoassay; tissue /cell culture; transfection; vesicle /vacuole; western blottings

DESCRIPTION (provided by applicant): Proteolytic processing of proenkephalin (PE) is required to produce active enkephalin opioid peptides that regulate analgesia, behavior, and immune cell function. It is, therefore, critical to define the multi-step proteolytic pathway required to convert PE into active enkephalin peptides. Our studies of PE processing have identified the cysteine protease termed 'prohormone thiol protease' (PTP) as the major PE cleaving activity in enkephalin-containing chromaffin granules. Notably, recent progress on this continuing project utilized active site affinity labeling and peptide microsequencing by mass spectrometry to identify the responsible PTP activity as cathepsin L. Cathepsin L is colocalized in secretory vesicles with enkephalin, and cathepsin L cleaves enkephalin-containing substrates at identical cleavage sites as native PTP. Significantly, cathepsin L knockout mice show reduced levels of enkephalin in brain. These new results implicate a key role for secretory vesicle cathepsin L in enkephalin peptide production. The cleavage specificities of cathepsin L and PTP generate peptide intermediates with NH2-terminal basic residue extensions. These findings indicate that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Indeed, Arg/Lys aminopeptidase activity is colocalized in chromaffin granules with PE, enkephalin, and PTP/cathepsin L. These new discoveries of cathepsin L and Arg/Lys aminopeptidase enyzmes for PE processing complement our earlier findings showing participation of the subtilisin-like PC1 and PC2 and carboxypeptidase E/H in processing PE. These findings provide the basis for the goal of this proposal that will investigate the roles of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, with PC1 and PC2, in the proteolytie pathway required for processing PE into enkephalin opioid peptides. This goal will be achieved in four specific aims, which will (1) evaluate PE processing by (a) determining the relative efficiency and cleavage sites for in vitro processing of PE by secretory vesicle cathepsin L, compared to PC1 and PC2, and (b) assessing cellular PE processing during enzyme coexpression with PE in PC12 cells, (2) assess the colocalization of cathepsin L with enkephalin and PC enzymes in chromaffin cells, transfected PC12 cells, and rat brain and neuroendocrine tissues, (3) examine the effects of reduced enzyme activity on PE processing in (a) chromaffin cells and cortical neurons subjected to antisense enzyme expression, as well as direct inhibition of cathepsin L with a selective chemical inhibitor, and in (b) cathepsin L knockout and PC2 knockout mice that show reduced enkephalin levels in brain, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for enkephalin production. Results can establish functional roles for two new protease components, secretory vesicle cathepsin L and Arg/Lys aminopeptidase, in the biosynthesis of enkephalin opioid peptides. These findings will enhance our knowledge of the complexity of the endogenous opioid system.

描述（由申请人提供）：需要对脑啡肽原（PE）进行蛋白水解加工来产生调节镇痛、行为和免疫细胞功能的活性脑啡肽阿片肽。因此，确定将 PE 转化为活性脑啡肽所需的多步蛋白水解途径至关重要。我们对 PE 加工的研究已确定，称为“激素原硫醇蛋白酶”(PTP) 的半胱氨酸蛋白酶是含脑啡肽嗜铬颗粒中主要的 PE 裂解活性。值得注意的是，该持续项目的最新进展利用活性位点亲和标记和质谱肽微测序来鉴定负责的 PTP 活性为组织蛋白酶 L。组织蛋白酶 L 与脑啡肽共定位于分泌囊泡中，并且组织蛋白酶 L 在与天然 PTP 相同的裂解位点处裂解含脑啡肽的底物。值得注意的是，组织蛋白酶 L 敲除小鼠大脑中的脑啡肽水平降低。这些新结果表明分泌囊泡组织蛋白酶 L 在脑啡肽生产中发挥着关键作用。组织蛋白酶 L 和 PTP 的裂解特异性产生带有 NH2 末端碱性残基延伸的肽中间体。这些发现表明，Arg/Lys 氨肽酶对于去除此类碱性残基是必需的。事实上，Arg/Lys 氨肽酶活性与 PE、脑啡肽和 PTP/组织蛋白酶 L 共定位于嗜铬颗粒中。组织蛋白酶 L 和 Arg/Lys 氨肽酶用于 PE 加工的这些新发现补充了我们之前的发现，即枯草杆菌蛋白酶样 PC1 和 PC2 以及羧肽酶 E/H 参与 PE 加工。这些发现为该提案的目标提供了基础，该提案将研究分泌囊泡组织蛋白酶 L 和精氨酸/赖氨酸氨肽酶以及 PC1 和 PC2 在将 PE 加工成脑啡肽阿片样肽所需的蛋白水解途径中的作用。这一目标将通过四个具体目标来实现，其中（1）通过以下方式评估 PE 加工：（a）与 PC1 和 PC2 相比，确定分泌囊泡组织蛋白酶 L 体外加工 PE 的相对效率和切割位点，以及（b）评估 PC12 细胞中酶与 PE 共表达期间的细胞 PE 加工，（2）评估组织蛋白酶 L 与脑啡肽和 PC 的共定位 (3) 在嗜铬细胞、转染的 PC12 细胞以及大鼠脑和神经内分泌组织中检测酶活性降低对 PE 加工的影响，其中 (a) 进行反义酶表达的嗜铬细胞和皮质神经元，以及用选择性化学抑制剂直接抑制组织蛋白酶 L，以及 (b) 组织蛋白酶 L 敲除和 PC2 敲除 显示大脑中脑啡肽水平降低的小鼠，以及 (4) 获得用于脑啡肽产生的 Arg/Lys 氨肽酶的生化和分子分析。结果可以确定两种新的蛋白酶成分（分泌囊泡组织蛋白酶 L 和精氨酸/赖氨酸氨肽酶）在脑啡肽阿片肽生物合成中的功能作用。这些发现将增强我们对内源性阿片类药物系统复杂性的了解。