Biosynthesis of Enkephalin Opioid Peptides

脑啡肽阿片肽的生物合成

基本信息

批准号：
7241534
负责人：
Vivian Y. H Hook
金额：
$ 29.3万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
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/7241534
关键词：
Absence of pain sensation Active Sites Adrenal Glands Adrenal Medulla Affinity Affinity Labels Aftercare Aminopeptidase Anabolism Analgesics Antisense Oligonucleotides Arginine Basic Amino Acids Behavior Biochemical Biological Assay Bos taurus Brain Cathepsin L Cattle Cell physiology Cells Chemicals Chromaffin Cells Chromaffin granule Cleaved cell Compatible Complement Complementary DNA Complex Condition Cysteine Protease Cytoplasmic Granules Data Drug or chemical Tissue Distribution Dynorphins Endopeptidases Endorphins Enkephalins Enzymes Evaluation Excision Family Funding Gel Chromatography Gene Silencing Goals Immune Immune Sera Immunoelectron Microscopy In Vitro Kinetics Knock-out Knockout Mice Knowledge Localized Lysine Mammalian Cell Mass Spectrum Analysis Mediating Methionine Enkephalin Microscopy Molecular Molecular Cloning N-terminal Neuroendocrine Cell Neurons Neuropeptides Neurosecretory Systems Opioid Opioid Peptide PC12 Cells Pain Pathway interactions Peptide Hydrolases Peptides Personal Satisfaction Physiologic pulse Plasmid Cloning Vector Plasmids Process Production Prohormone Convertase Property Proprotein Convertase 1 Proprotein Convertase 2 Protein Precursors Proteins Proteolytic Processing Pulse taking Radioimmunoassay Rattus Recombinants Regulation Relative (related person)Rodent Role Secretory Vesicles Site Specificity Substrate Specificity Subtilisin Subtilisins System Testing Time Tissues Vaccinia Vaccinia virus Viral Western Blotting affinity labeling aminopeptidase B arginyllysine base brain tissue carboxypeptidase H endogenous opioids enkephalin degrading enzyme enzyme activity in vivo inhibitor/antagonist interest mRNA Expression member proenkephalin prohormone thiol protease protein aminoacid sequence protein expression

项目摘要

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）：pro骨（PE）的蛋白水解处理需要产生调节镇痛，行为和免疫细胞功能的活性enkephalin阿片类肽。因此，定义将PE转换为活跃的Enkephalin肽所需的多步蛋白水解途径至关重要。我们对PE加工的研究确定了称为“激素硫醇蛋白酶”（PTP）的半胱氨酸蛋白酶在含Enkephalin的铬脂蛋白颗粒中是主要的PE切割活性。值得注意的是，这个持续项目的最新进展利用了主动部位亲和力标记和质谱的肽测序，将负责任的PTP活性确定为calter蛋白蛋白酶L. calterpsin l. calterepsin l calter蛋白L.在分泌囊泡中与enkephalin和cantepsin l cleaves cankephalin conkephalin cenkephalin conkephalin cenkephalins contes celltreatials clates senes serates senes as sectes sates as avage sedpp。值得注意的是，组织蛋白酶L基因敲除小鼠显示大脑中的脑脑水平降低。这些新结果暗示了分泌囊泡组织蛋白酶L在邻齿肽产生中的关键作用。组织蛋白酶L和PTP的切割特异性与NH2末端基本残基扩展产生肽中间体。这些发现表明，ARG/LYS氨基肽酶是必要的，以去除此类基本残基。实际上，ARG/LYS氨基肽酶的活性在铬蛋白颗粒中与PE，Enkephalin和PTP/Catherepsin L.共定位。这些新发现的组织蛋白酶L和ARG/Lys氨基肽酶Enyzmes的PE处理以补充了我们的较早的PC2和PC2的参与，以补充PE的PE处理。这些发现为该提案的目标提供了基础，该目标将研究分泌囊泡组织蛋白酶L和ARG/LYS氨基肽酶，具有PC1和PC2的作用，在将PE加工到Enkephalin阿片类肽中所需的Proteolytie途径中。该目标将在四个具体目标中实现，（1）通过（a）通过（a）确定与PC1和PC1和PC1和PC1和（b）评估PC12细胞（2）Collocal（2）Collocal（2）Collocal（2）Collocal（2）Collocal（2）Collocal collocal（2）Collocal（2）Collocal在PC1和PC1中相比，通过分泌囊泡组织L相比，确定通过分泌囊泡组织蛋白酶L的相对效率和切割位点进行体外处理的相对效率和切割位点，并评估PE处理。（3）检查酶活性降低对（a）（a）（a）铬脂蛋白细胞和皮质神经元中PE加工对PE加工的影响（3），在（抗敏感性的抗敏感性表达，以及抑制Cathepsin lokection noknitive noknitive nockitive noknitive innkitor and officige）（a））检查（3）显示出脑中脑脑水平降低的小鼠，以及（4）获得ARG/LYS氨基肽酶的生化和分子分析以进行nkephalin的产生。结果可以在enkephalin阿片类肽的生物合成中为两个新的蛋白酶成分（分泌囊泡结肠L和ARG/LYS氨基肽酶）建立功能作用。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。