POMC Processing and Beta-Endorphine-Related Opioid Peptides

POMC 加工和 β-内啡肽相关阿片肽

• 批准号: 7816915
• 负责人: Vivian Y. H Hook
• 金额: $ 31.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7816915
• 关键词: Absence of pain sensation; Active Sites; Affinity; Affinity Labels; Aminopeptidase; Anabolism; Anterior Pituitary Gland; Antisense Oligonucleotides; Basic Amino Acids; Behavior; Biochemical; Biological Assay; Brain; Brain region; CLIK148; Cathepsin L; Cattle; Cell Line; Cells; Chemicals; Cleaved cell; Confocal Microscopy; Corticotropin; Cultured Cells; Cysteine Protease; Cysteine Proteinase Inhibitors; Data; Drug or chemical Tissue Distribution; Endorphins; Enzymes; Evaluation; Family; Genes; Goals; Immune Sera; Immunoelectron Microscopy; In Vitro; Kinetics; Knock-out; Knockout Mice; Knowledge; Lead; Mammalian Cell; Mass Spectrum Analysis; Measurement; Microscopy; Molecular; Molecular Cloning; Mus; N-terminal; Neuroendocrine Cell; Neurons; Neuropeptides; Neurosecretory Systems; Opioid Peptide; Pain; Pathway interactions; Peptide Hydrolases; Peptides; Physiologic pulse; Physiological; Pituitary Gland; Plasmids; Primary Cell Cultures; Pro-Opiomelanocortin; Process; Production; Prohormone Convertase; Property; Proprotein Convertase 1; Proprotein Convertase 2; Protease Gene; Protein Precursors; Proteolysis; Proteolytic Processing; Rattus; Recombinants; Relative (related person); Role; Secretory Vesicles; Side; Site; Small Interfering RNA; Specificity; Stress; Substrate Specificity; Subtilisins; System; Vaccinia; Vesicle; Viral; Western Blotting; affinity labeling; aminopeptidase B; arginyllysine; base; beta-Endorphin; brain cell; brain tissue; carboxypeptidase H; comparative; endogenous opioids; enzyme activity; in vivo; inhibitor/antagonist; knockout gene; meetings; member; mind control; molecular sieving; novel; peptide B; peptide hormone; prohormone; protease inhibitor E64d; protein aminoacid sequence; remediation; research study; secretion process

DESCRIPTION (provided by applicant): The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems.

描述（由申请人提供）：内源性β-内啡肽阿片肽的生物合成需要对其POMC（阿黑皮质素原）前体进行蛋白水解加工。 β-内啡肽是镇痛、行为和压力的关键调节剂。因此，定义将 POMC 转化为活性 β-内啡肽所需的蛋白水解途径至关重要。基于组织蛋白酶 L 敲除小鼠的令人兴奋的新结果（显示 β-内啡肽水平降低），我们的研究已确定分泌囊泡组织蛋白酶 L 是 POMC 的关键加工酶。这些新颖的结果是通过从分泌囊泡中纯化、活性位点亲和标记和肽微测序来鉴定组织蛋白酶 L 的加工活性而获得的。组织蛋白酶 L 定位于含有 POMC 的分泌囊泡以及含有神经肽的分泌囊泡。这些新结果表明分泌囊泡组织蛋白酶 L 在 β-内啡肽和神经肽的产生中发挥着重要作用。组织蛋白酶 L 对二碱基加工位点的裂解特异性产生具有 NH2 末端碱性残基的肽中间体，表明需要 Arg/Lys 氨肽酶来去除此类碱性残基。精氨酸/赖氨酸氨肽酶活性与 β-内啡肽和神经肽共定位于神经分泌小泡中。这些新结果表明，除了众所周知的枯草杆菌蛋白酶样 PC1 和 PC2 以及羧肽酶 E/H 途径之外，组织蛋白酶 L 和 Arg/Lys 氨肽酶也是激素原加工的新蛋白酶途径。因此，本提案的目标是确定与 PC1 和 PC2 相比，分泌囊泡组织蛋白酶 L 和 Arg/Lys 氨肽酶在将 POMC 加工成 β-内啡肽方面的作用。这一目标将通过四个具体目标来实现，即 (1) 确定 (a) 组织蛋白酶 L 敲除小鼠与 PC1 和 PC2 敲除小鼠相比，垂体和大脑中酶活性降低对 POMC 加工的影响，(b) siRNA 实验为了降低垂体细胞和脑神经元细胞中的酶水平，以及在组织蛋白酶 L 的直接化学抑制实验中，(2) 评估组织蛋白酶 L 与 β-内啡肽和 PC 酶在分泌中的定位(3) 通过将每种酶与 POMC 共转染，确定每种加工酶在 (a) PC12 神经内分泌细胞（和 GH3 细胞）中的细胞 POMC 加工中的作用，以及 (b) 体外动力学和裂解中的作用POMC 加工的现场研究，(4) 获得用于 β-内啡肽生产的 Arg/Lys 氨肽酶的生化和分子分析。结果将确定分泌囊泡组织蛋白酶 L 和精氨酸/赖氨酸氨肽酶加工途径在 β-内啡肽生物合成中的作用。该项目的新发现将增强我们对内源性阿片类药物和神经肽系统生物合成机制复杂性的了解。