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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）：需要对前脑啡肽（proenkephalin， PE）进行蛋白水解处理，以生产调节镇痛、行为和免疫细胞功能的活性脑啡肽类阿片肽。因此，确定将PE转化为活性脑啡肽所需的多步骤蛋白水解途径至关重要。我们对PE加工的研究已经确定了半胱氨酸蛋白酶称为“激素原硫醇蛋白酶”（PTP）是含脑啡肽的染色质颗粒中主要的PE切割活性。值得注意的是，这个持续项目的最新进展利用活性位点亲和标记和肽微测序的质谱鉴定了PTP的活性与组织蛋白酶L有关。组织蛋白酶L与脑啡肽共定位在分泌囊泡中，组织蛋白酶L在与天然PTP相同的切割位点上切割含有脑啡肽的底物。值得注意的是，组织蛋白酶L敲除小鼠显示脑啡肽水平降低。这些新的结果暗示了分泌囊泡组织蛋白酶L在脑啡肽产生中的关键作用。组织蛋白酶L和PTP的切割特性产生具有nh2末端碱性残基延伸的肽中间体。这些发现表明精氨酸/赖氨酸氨基肽酶是去除这些碱性残基所必需的。事实上，精氨酸/赖氨酸氨基肽酶活性在染色质颗粒中与PE、脑啡肽和PTP/组织蛋白酶L共定位。这些组织蛋白酶L和精氨酸/赖氨酸氨基肽酶参与PE加工的新发现补充了我们先前的发现，即枯草杆菌样PC1和PC2以及羧肽酶E/H参与PE加工。这些发现为研究分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨基肽酶以及PC1和PC2在将PE加工成脑啡肽类阿片肽所需的蛋白水解途径中的作用提供了基础。这一目标将通过四个具体目标来实现，即：(1)通过(a)确定与PC1和PC2相比，分泌囊泡组织蛋白酶L在体外处理PE的相对效率和切割位点来评估PE加工，(b)评估PC12细胞中与PE酶共表达时的细胞PE加工，(2)评估组织蛋白酶L与脑啡肽和PC酶在染色质细胞、转染的PC12细胞和大鼠脑和神经内分泌组织中的共定位。(3)检测酶活性降低对(a)受反义酶表达的染色质细胞和皮质神经元PE加工的影响，以及选择性化学抑制剂对组织蛋白酶L的直接抑制，以及(b)组织蛋白酶L敲除和PC2敲除显示脑啡肽水平降低的小鼠，以及(4)获得脑啡肽产生的精氨酸/赖氨酸氨基肽酶的生化和分子分析。结果表明，分泌囊泡组织蛋白酶L和精氨酸/赖氨酸氨基肽酶两种新的蛋白酶组分在脑啡肽类阿片肽的生物合成中具有功能作用。这些发现将增强我们对内源性阿片系统复杂性的认识。