NEURAL EP24.15-A MODEL FOR NEUROPEPTIDASE FUNCTION

描述(改编自申请人的摘要)： 本应用程序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/调节神经肽的内肽酶家族。EP24.15是 存在于大脑、脑下垂体和性腺中，并直接与 关键生理通路与神经内分泌/神经退行性疾病 和癌症。这种酶在细胞外处理神经肽，因此 在调节细胞生长所必需的信号转导事件和 差异化。了解EP24的功能。15及其相互作用 底物和抑制剂将提供对以下方面的独特见解 正常和疾病状态，并将有助于设计新的治疗药物 治疗各种疾病。此应用程序中的实验将集成 生化、遗传学、理论、结构和细胞生物学方法 研究了该金属酶的酶-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 本项目的具体目标是：具体目标1.要素的确定 负责EP24.15 EP24中的底物绑定和编目。15蛋白质 将利用结构遗传学和动力学进行功能表征 决定。瞄准啦。是什么氨基酸阻断剂导致了 神经肽的催化作用？瞄准磅。底物由哪些残基组成[和 抑制物]酶的结合亚基？具体目标2.定义组件 调制rEP24。15个活跃度。本地化。和衬底尺寸的选择性。 4.15在体外/体内被磷酸化，存在于细胞核中，尽管 内寡肽酶，能裂解一些大底物。 目标2a。确定磷酸化对EP24的影响。15酶活性。 目标2b。转移它的酶的结构决定因素是什么？ 到原子核吗？ 目标2c。EP24。15被定义为“内寡肽酶”。其机制是什么？ 对大小有选择性？ 特定的目标3。EP24的模拟和高分辨率研究。15个结构 生物学方法：同源结构域和衍射的分子模拟 分析。目标3a。从细菌同源物为这一类建立一个原型 哺乳动物的酶。目标3b。实施多波长反常衍射 对EP24的分析。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.