EXTRACELLULAR NEUROPEPTIDE PROCESSING ENDOPEPTIDASE

细胞外神经肽加工内肽酶

基本信息

批准号：
6447379
负责人：
JAMES L. ROBERTS
金额：
$ 18.81万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-02-08 至 2003-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6447379
关键词：
cell membrane enzyme activity enzyme linked immunosorbent assay enzyme structure extracellular matrix gene expression gene mutation isozymes messenger RNA metalloendopeptidases neuropeptides nucleic acid sequence phosphorylation ribosomes secretory protein

项目摘要

Neuropeptides are important in transducing changes through their interaction with receptors in the extracellular milieu. Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble metallopeptidase with broad expression in the brain that is involved in neuropeptide metabolism. Enzymes like EP24.15 either: inactivate peptides, convert an inert precursor to an active moiety, or in some instances, convert on bioactive peptide into another with different agonist properties. While the soluble, cytosolic forms of these enzymes have been characterized in purified form, including EP24.15, the properties of the secreted enzyme, much less the secretory mechanisms for the enzymes, have not been extensively studied. Thus, it is important to characterize the enzyme with respect to targeting to the extracellular milieu and the factors modulating enzyme activity extracellularly. This will be accomplished in three Specific Aims. Specific Aim 1: How is EP 24.15 enzyme targeted to the extracellular milieu? Aim 1a. Are there different forms of the EP24.15 derived from the same or different MRNAs? Are the membrane-bound and free ribosome mRNA forms different? What is the structure of the EP24.15 encoding gene? Aim 1b. Determine the secretory pathways targeting the secreted and plasma membrane forms of EP24.15 Which secretory pathway for EP24.15 is subserved by the individual EP24.15 mRNAs? Does mutation of the phosphorylation site and/or cysteinyl mutants change EP24.15 release? Specific Aim 2: Characterize the association between EP24.15 and the plasma membrane or extracellular matrix (ECM). Aim 2a. What is the biochemical differences between plasma membrane and soluble EP24.15? Is the plasma membrane/media form of EP24.15 proteolytically processed? How is EP24.15 associated with the plasma membrane? Aim 2b. Which specific ECM components bind EP24.15 and which EP24.15 are involved in the interaction(s)? Aim 2c. What are the effects of phosphorylation and redox status on EP24.15's association with the extracellular environment? Is the amino terminal domain of EP24.15 important in this association? Specific Aim 3: Determine whether EP24.15 interactions with the cell surface of ECM can modulate EP24.15 activity. Aim 3a. Are the changes in EP24.15 redox-dependent multimerization between different extracellular compartments? Do the mutations which abolish thiol activation of the recomb. enzyme similarly affect the extracellular pool of EP24.15? Aim 3b. Does phosphorylation alter the activity/partition of EP24.15 in the extracellular environment? Determine which form of EP24.15 and the residue on the EP24.15 protein for phosphorylation in vivo.

神经肽在通过其转导变化方面发挥着重要作用与细胞外环境中的受体相互作用。内肽酶EC 3.4.24.15 (EP24.15) 是一种可溶性金属肽酶，广泛表达于参与神经肽代谢的大脑。酶类如 EP24.15 要么：使肽失活，将惰性前体转化为活性部分，或在某些情况下，将生物活性肽转化为另一种具有不同的激动剂特性。而可溶性、细胞质这些酶的形式已被表征为纯化形式，包括 EP24.15，分泌酶的特性，更不用说分泌了酶的作用机制尚未得到广泛研究。因此，它是重要的是表征酶的靶向性细胞外环境和酶活性调节因素细胞外。这将通过三个具体目标来实现。具体目标 1：EP 24.15 酶如何靶向细胞外环境？目标 1a。 EP24.15 是否有源自相同或相同形式的不同形式？不同的mRNA？ mRNA有膜结合型和游离型两种形式不同的？ EP24.15编码基因的结构是怎样的？目标 1b。确定针对分泌物和血浆的分泌途径 EP24.15 的膜形式 EP24.15 的分泌途径受到影响由各个 EP24.15 mRNA 决定？磷酸化位点是否突变和/或半胱氨酰突变体会改变 EP24.15 的发布吗？具体目标 2：描述 EP24.15 与质膜或细胞外基质 (ECM)。目标 2a。质膜和质膜之间的生化差异是什么可溶EP24.15？是EP24.15的质膜/介质形式蛋白水解加工？ EP24.15 与等离子体有何关联膜？目标 2b。哪些特定 ECM 成分结合 EP24.15，哪些 EP24.15 是参与互动？目标 2c。磷酸化和氧化还原状态对细胞有什么影响 EP24.15 与细胞外环境的关联？是氨基吗 EP24.15 的终端域在此关联中重要吗？具体目标 3：确定 EP24.15 是否与细胞相互作用 ECM表面可以调节EP24.15活性。目标 3a。 EP24.15 氧化还原依赖性多聚化的变化是不同的细胞外区室？做废除硫醇的突变重组的激活。酶同样影响细胞外池 EP24.15？目标 3b。磷酸化是否会改变 EP24.15 的活性/分配细胞外环境？确定 EP24.15 的哪种形式以及 EP24.15 蛋白上的残基用于体内磷酸化。