Proteolytic Mechanisms for Production of the Dynorphin Peptide Neurotransmitter

产生强啡肽神经递质的蛋白水解机制

• 批准号: 7612823
• 负责人: ARDALAN MINOKADEH
• 金额: $ 2.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7612823
• 关键词: Absence of pain sensation; Adrenal Glands; Adrenal Medulla; Anabolism; Brain; Brain region; CLIK148; CTSL gene; Cathepsin L; Cell Extracts; Cells; Chromaffin Cells; Chromogranin A; Cleaved cell; Complementary DNA; Confocal Microscopy; Cysteine; Cysteine Protease; Data; Drug abuse; Dynorphin A; Dynorphins; Endorphins; Enkephalins; Evaluation; Gene Expression; Goals; Immune Sera; Immunofluorescence Immunologic; Knock-out; Knockout Mice; Knowledge; Mass Spectrum Analysis; Measures; Mediating; Mus; Neuroendocrine Cell; Neuronal Plasticity; Neurons; Neuropeptides; Neurosecretory Systems; Neurotransmitters; Nicotine; Opioid; Opioid Peptide; PC12 Cells; Pain; Pathway interactions; Peptide Hydrolases; Peptides; Pituitary Gland; Play; Process; Production; Prohormone Convertase; Proprotein Convertase 1; Proprotein Convertase 2; Proprotein Convertases; Protease Gene; Protein Precursors; Proteolysis; Proteolytic Processing; Radioimmunoassay; Relative (related person); Role; Secretory Vesicles; Structure; Substance abuse problem; Subtilisins; System; Tertiary Protein Structure; Tissues; Western Blotting; base; chronic pain; in vivo; inhibitor/antagonist; novel; prodynorphin; research study; rimorphin

DESCRIPTION (provided by applicant): The opioid peptide neurotransmitter dynorphin is synthesized through the proteolysis of the larger prodynorphin precursor protein. Dynorphin has a well-documented role in modulating pain and mechanisms involved in substance abuse. A better understanding of these important processes could therefore emerge from elucidation of the proteolytic pathways through which inactive prodynorphin is cleaved to produce active dynorphin peptides. Two primary protease pathways are implicated in the synthesis of mature peptide neurotransmitters, or neuropeptides, consisting of the recently characterized cathepsin L cysteine protease in secretory vesicles, together with the well-known proprotein convertase (PC) proteases PC 1/3 and PC2. New results demonstrate participation of the novel cathepsin L cysteine protease pathway in secretory vesicles for dynorphin biosynthesis. Notably, cathepsin L gene knockout mice show that dynorphin levels in brain are reduced by 75%. Furthermore, expression of cathepsin L results in dynorphin production in PC12 cells. Based on these new findings, it is hypothesized that cathepsin L is an important processing protease for dynorphin production. The goal of this proposal is to evaluate the production of dynorphin from prodynorphin by cathepsin L This goal will be achieved in three specific aims. Specific aim 1 will assess dynorphin peptide production by cathepsin L during coexpression of prodynorphin and cathepsin L in neuroendocrine PC12 cells, combined with cellular evaluation of cathepsin L colocalization with dynorphin in secretory vesicles of the regulated secretory pathway. Complementary in vivo studies of cathepsin L will be achieved in specific aim 2 which will assess dynorphin and prodynorphin-derived products in cathepsin L gene knockout mice, with comparisons to PC1/3 and PC2 knockout mice. Results will indicate the impact of each of these processing proteases on dynorphin neuropeptide production. Specific aim 3 will assess the effects of a selective inhibitor of cathepsin L during dynorphin production in chromaffin cells and brain cortical neurons in primary culture. Results will indicate how cellular systems utilize the cathepsin L processing pathway for production of dynorphin. Dynorphin plays an important role in modulating pain, including chronic pain, and neuroplasticity associated with drug abuse. Knowledge gained from this study will increase our understanding of these processes and provide targets for regulatory agents that may modulate dynorphin production to facilitate its beneficial effects or reduce its adversive effects.

描述（由申请人提供）：阿片肽神经递质dynorphin是通过较大的prodynorphin前体蛋白的蛋白水解合成的。Dynorphin在调节疼痛和药物滥用机制方面有充分的证据。因此，对这些重要过程的更好理解可以从蛋白质水解途径的阐明中出现，通过该途径，无活性的前肌啡被裂解为产生活性的肌啡肽。两种主要的蛋白酶途径与成熟多肽神经递质或神经肽的合成有关，包括最近在分泌囊泡中发现的组织蛋白酶L半胱氨酸蛋白酶，以及众所周知的蛋白转化酶（PC）蛋白酶pc1 /3和PC2。新的研究结果表明，新的组织蛋白酶L半胱氨酸蛋白酶途径参与分泌囊泡的肌啡肽生物合成。值得注意的是，组织蛋白酶L基因敲除小鼠显示，大脑中的肌啡水平降低了75%。此外，组织蛋白酶L的表达导致PC12细胞产生促啡肽。基于这些新发现，我们推测组织蛋白酶L是一种重要的促啡肽生产加工蛋白酶。本提案的目标是评估组织蛋白酶L从原肌啡生成肌啡。该目标将在三个具体目标中实现。具体目的1将评估组织蛋白酶L在神经内分泌PC12细胞中原肌啡肽和组织蛋白酶L共表达时产生的肌啡肽，并结合组织蛋白酶L与肌啡肽在调节分泌途径的分泌囊泡中共定位的细胞评价。组织蛋白酶L的补充体内研究将在特定目标2中实现，该目标将评估组织蛋白酶L基因敲除小鼠体内的肌啡肽和前肌啡肽衍生产物，并与PC1/3和PC2敲除小鼠进行比较。结果将表明这些加工蛋白酶对肌啡肽神经肽产生的影响。特异性目的3将评估组织蛋白酶L的选择性抑制剂在原代培养的嗜铬细胞和脑皮质神经元中产生强啡肽的作用。结果将表明细胞系统如何利用组织蛋白酶L加工途径来生产肌啡。Dynorphin在调节疼痛（包括慢性疼痛）和与药物滥用相关的神经可塑性方面发挥重要作用。从这项研究中获得的知识将增加我们对这些过程的理解，并为调节肌啡生成以促进其有益作用或减少其不利影响的调节剂提供靶标。