EXTRACELLULAR NEUROPEPTIDE PROCESSING ENDOPEPTIDASE

神经肽在通过其转导变化方面起着重要作用 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。像这样的酶 EP24.15或：灭活多肽，将惰性前体转化为 活性部分，或在某些情况下，将生物活性多肽转化为 另一种具有不同的激动剂特性。而可溶性的、胞质的 这些酶的形式已经以纯化形式进行了表征，包括 EP24.15，分泌酶的性质，更不是分泌酶的性质 这种酶的作用机制还没有得到广泛的研究。因此，它是 重要的是根据靶向于 细胞外环境及其影响酶活性的因素 在细胞外。这将通过三个具体目标来实现。 特定目标1：EP 24.15酶如何靶向胞外 环境？ 目标1a。是否有不同形式的EP24.15源自相同或 不同的mRNA？是膜结合核糖体和游离核糖体吗？ 不一样？EP24.15编码基因的结构是什么？ 目标1b。确定针对分泌物和血浆的分泌途径 支持EP24.15分泌途径的膜形式 由单个EP24.15 mRNAs？磷酸化位点的突变 和/或半胱氨酸基突变改变EP24.15的释放？ 具体目标2：确定EP24.15和 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？ 可溶性EP24.15？是EP24.15的质膜/介质形式吗？ 经过蛋白质分解处理的？EP24.15与血浆有何关联 薄膜？ 目标2b。哪些特定的ECM组件绑定EP24.15，哪些是EP24.15 参与互动(S)？ 目标2c。磷酸化和氧化还原状态对 EP24.15‘S与细胞外环境的关系？是氨基吗 EP24.15的末端结构域在这种联系中重要吗？ 具体目标3：确定EP24.15是否与细胞相互作用 ECM表面对EP24.15活性有调节作用。 目标3a。EP24.15依赖氧化还原的多聚化在 不同的细胞外隔间？使硫醇消失的突变 重组梳子的激活。酶同样会影响细胞外池 EP24.15？ 目标3b。磷酸化是否改变EP24.15的活性/分配 细胞外环境？确定EP24.15的哪种形式和 EP24.15蛋白上的残基用于体内磷酸化。

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.