NEURAL EP24.15-A MODEL FOR NEUROPEPTIDASE FUNCTION

神经肽酶功能的神经 EP24.15-A 模型

• 批准号: 6548649
• 负责人: Marc J Glucksman
• 金额: $ 15.6万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-20 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6548649
• 关键词: X ray crystallography; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; neuropeptides; peptidases; phosphorylation; protein localization; protein sequence; site directed mutagenesis

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.

描述（改编自申请人的摘要）： 本申请的总体目标是阐明生物化学 哺乳动物原型成员 E.C.3.4.24.15 [EP24.15] 的功能 处理/调节神经肽的肽链内切酶家族。 EP24.15是 存在于大脑、垂体和性腺中，并与 关键生理途径和神经内分泌/神经退行性疾病 和癌症。这种酶在细胞外加工神经肽，因此 对于调节细胞生长所必需的信号转导事件至关重要 差异化。了解EP24的功能。 15 及其相互作用 与底物和抑制剂的结合将为各方面提供独特的见解 正常和疾病状态，将有助于设计新的治疗剂 用于多种疾病。该应用程序中的实验将集成 生物化学、遗传、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 也适用于其他大分子。因此，相互关联的 该项目的具体目标是： 具体目标 1. 元素的确定 EP24.15 EP24 中负责底物结合和催化。 15 蛋白质 将利用结构遗传学和动力学进行功能表征 的决定。瞄准啦。哪些氨基酸决定因素负责 神经肽的催化作用？目标 lb. 哪些残基构成底物 [和 抑制剂]酶的结合亚位点？具体目标 2. 定义组件 调制器 EP24。 15 活动.本土化。和基材尺寸选择性。 4.15 在体外/体内被磷酸化，存在于细胞核中，并且通过 寡肽内切酶，可以切割一些大的底物。 目标 2a。确定磷酸化对 EP24 的影响。 15酶活性。 目标 2b。易位酶的结构决定因素是什么 到核？ 目标 2c。 EP24。 15被定义为“寡肽内切酶”。机制是什么 尺寸选择性？ 具体目标3。 EP24 的建模和更高分辨率的研究。 15 结构 生物学方法：同源域的分子建模和衍射 分析。目标 3a。从细菌同系物构建此类的原型 哺乳动物酶。目标 3b。执行多波长反常衍射 EP24 的分析。 15.