NEURAL EP24.15-A MODEL FOR NEUROPEPTIDASE FUNCTION

描述（改编自申请人摘要）： 本申请的总体目标是阐明生物化学过程。 E.C.3.4.24.15 [EP24.15]的功能，哺乳动物的原型成员 加工/调节神经肽的内肽酶家族。EP24.15是 存在于大脑，垂体和性腺中，并直接涉及 关键生理通路和神经内分泌/神经退行性疾病 和癌症这种酶在细胞外加工神经肽， 在调节细胞生长所必需的信号转导事件中至关重要， 分化了解EP 24的功能。15、相互作用 与底物和抑制剂将提供独特的洞察方面， 并将有助于设计新的治疗剂 治疗各种疾病本应用中的实验将整合 生物化学、遗传学、理论、结构和细胞生物学方法， 研究这种金属酶的酶-配体相互作用。这些策略 也适用于其他高分子。因此，相互关联的 该项目的具体目标是：具体目标1。元素测定 在EP 24.15和EP 24中负责底物结合和催化。15蛋白 将利用结构遗传学和动力学进行功能表征 决心。瞄准洛杉矶。什么氨基酸决定了 神经肽的催化作用瞄准磅。哪些残基构成底物[和 抑制剂]结合酶的亚位点？具体目标2。定义组件 调制器EP 24. 15活跃。本地化和衬底尺寸选择性。 4.15在体外/体内被磷酸化，存在于细胞核中， 内切寡肽酶，可以切割一些大的底物。 目标2a。确定磷酸化对EP 24的影响。15酶活性 目标2b。什么是这种酶的结构决定因素， 到原子核？ 目标2c。EP24. 15定义为“内寡肽酶”。是什么机制 大小选择性？ 具体目标3。EP 24的建模和更高分辨率研究。15结构 生物学方法：同源结构域和衍射的分子模拟 分析。目标3a。从细菌同源物中为这类 哺乳动物的酶目标3b。执行多波长异常衍射 分析EP 24。15.

DESCRIPTION(Adapted from applicant's abstract): The overall objective of this application is to elucidate the biochemical functions of E.C.3.4.24.15 [EP24.15], the prototypical member of the mammalian family of endopeptidases that process/modulate neuropeptides. EP24.15 is present in brain, pituitary, and gonads, and has been directly implicated in critical physiologic pathways and neuroendocrine/neurodegenerative disorders and cancer. This enzyme processes neuropeptides extracellularly and thus is crucial in regulating signal transduction events essential to cell growth and differentiation. Understanding the function of EP24. 15 and its interaction with substrates and inhibitors will provide unique insight into aspects of normal and disease states and will aid in the design of new therapeutic agents for a variety of disorders. Experiments in this application will integrate biochemical, genetic, theoretical, structural and cell biological approaches to studying the enzyme-ligand interactions of this metalloenzyme. These strategies are also applicable to other macromolecules. Therefore, the interrelated specific aims of this project are: Specific Aim 1. Determinatlon of elements responsible for substrate binding and catalvsis in EP24.15 EP24. 15 protein will be functionally characterized utilizing structural genetics and kinetic determinations. Aim la. What amino acid deterrninants are responsible for the catalysis of neuropeptides? Aim lb. Which residues comprise the substrate [and inhibitor] binding subsites of the enzyme? Specific Aim 2. Define components modulatingr EP24. 15 activitv. localization. and substrate size selectivity. 4.15 is phosphorylated in vitro/in vivo, present in the nucleus, and though an endooligopeptidase, can cleave some large substrates. Aim 2a. Determine the effect of phosphorylation upon EP24. 15 enzyme activity. Aim 2b. What are the structural determinants of the enzyme which translocate it to the nucleus? Aim 2c. EP24. 15 is defined as an 'endooligopeptidase'. What is the mechanism of size selectivity? Specific Aim3. Modeling and higher resolution studies of EP24. 15 Structural biological approaches: molecular modeling of homologous domains and diffraction analyses. Aim 3a. Build a prototype from bacterial homologues for this class of mammalian enzymes . Aim 3b. Execute multiwavelength anomalous diffraction analyses of EP24. 15.