EXTRACELLULAR NEUROPEPTIDE PROCESSING ENDOPEPTIDASE

神经肽在通过其传导变化中是重要的。 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，在大肠杆菌中广泛表达。 参与神经肽代谢的大脑。酶如 EP24.15或者：肽类，将惰性前体转化为 活性部分，或在某些情况下，在生物活性肽上转化成 另一种具有不同的激动剂特性。而可溶性，胞质 这些酶的形式已经以纯化形式表征，包括 EP24.15，分泌酶的性质，更不用说分泌酶了。 酶的作用机制尚未被广泛研究。照经上所 重要的是表征酶的靶向， 细胞外环境及酶活性调节因子 在细胞外。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。是否有不同形式的EP24.15源自相同或 不同的MRNA？是膜结合和游离核糖体mRNA形式 不一样吗EP24.15编码基因的结构是什么？ 目标1b。确定靶向分泌和血浆的分泌途径 EP 24.15的膜形式，其分泌途径受影响 EP24.15 mRNA的表达磷酸化位点的突变 和/或半胱氨酰突变体改变EP24.15释放？ 具体目标2：描述EP 24.15与 质膜或细胞外基质（ECM）。 目标2a。什么是生物膜之间的生化差异和 可溶性EP 24.15？是EP 24.15的质膜/培养基形式 蛋白水解处理的EP24.15如何与血浆相关 膜？ 目标2b。哪些特定ECM组件与EP 24.15绑定，哪些EP 24.15是 参与互动（S）？ 目标2c。磷酸化和氧化还原状态对 EP24.15与细胞外环境的关系？是氨基 EP24.15的末端结构域在这种关联中重要吗？ 具体目标3：确定EP 24.15是否与细胞相互作用 ECM表面可调节EP 24.15活性。 目标3a。EP24.15氧化还原依赖性多聚化的变化是否与 不同的细胞外区室那些废除巯基的突变 激活recomb。酶类似地影响细胞外池 EP24.15？ 目标3b。磷酸化是否改变了EP24.15的活性/分配？ 细胞外环境？确定EP24.15的哪种形式， 15蛋白上的残基进行体内磷酸化。

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.