Biochemistry and Physiology of Peptide Amidation

肽酰胺化的生物化学和生理学

基本信息

批准号：
8435472
负责人：
ELIZABETH ANNE EIPPER
金额：
$ 53.47万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-06-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8435472
关键词：
AQP1 gene Affect Amides Amygdaloid structure Appearance Behavioral Biochemistry Biological Biology C-terminal Cell Nucleus Cells Cnidaria Communication Copper Crystallization Cues Cytoplasmic Tail Cytosol Data Diet Dietary Copper Electrophysiology (science)Endocrine Endocrine system Enzymatic Biochemistry Enzymes Event Evolution Exhibits Exopeptidase Gene Expression Gene Targeting Generations Genes Genetic Genetic Transcription Glutamates Goals Homeostasis Hormones Human Improve Access Knowledge Leukocytes Lyase Measures Membrane Metabolism Methods Microarray Analysis Mixed Function Oxygenases Modification Molecular Molecular Chaperones Multivesicular Body Mus Mutation Nervous system structure Neuropeptides Nuclear Oxygen Pathway interactions Peptides Phosphorylation Physiologic Thermoregulation Physiological Physiological Processes Physiology Pituitary Gland Pituitary Hormones Play Production Proprotein Convertase 2 Protease Inhibitor Protein Isoforms Proteins Proteolysis RNA Splicing Reliance Role Sea Anemones Secretory Vesicles Signal Transduction Sodium Chloride Structure Synapses System Testing Variant Vasopressins Work X-Ray Crystallography adrenomedullin amidation antileukoprotease ascorbate base behavior observation behavior test carboxypeptidase H cofactor cytochrome b561 human SLPI protein hypocupremia improved intercellular communication man mutant neoplastic cell peptidylglycine alpha-amidating monooxygenase planetary Atmosphere preprohormone public health relevance response trafficking vasoconstriction

项目摘要

DESCRIPTION (provided by applicant): Peptides play essential roles throughout the endocrine and nervous systems. The biosynthetic pathway leading from preprohormone to product peptide is fundamentally similar in human and in primitive creatures such as the sea anemone. As precursors move through the lumen of the secretory pathway, endoproteases, exopeptidases and peptidylglycine 1-amidating monooxygenase (PAM), the enzyme responsible for crucial C- terminal amidation, function sequentially. Peptidylglycine 1-hydroxylating monooxygenase (PHM; EC1.14.17.3), the first enzyme of the bifunctional PAM protein, requires copper and ascorbate; in man, both must be acquired from the diet. Cuproenzymes like PHM, rare in anaerobic species, evolved with the advent of molecular oxygen in the atmosphere and are overwhelmingly associated with its use. Our analyses of mice with one functional PAM gene (PAM mice) revealed multiple physiological and behavioral alterations with slight decreases in the amidated peptides measured. Many of the alterations were ameliorated by additional dietary copper and mimicked in copper deficient wildtype mice, leading to the conclusion that PAM plays a role in copper homeostasis. We will build on our discovery that membrane PAM yields a soluble fragment of its cytosolic domain (sfCD) that is targeted to the nucleus and alters gene expression, to determine the mechanisms through which PAM affects physiological function. Aim 1: The structures determined for PHM and peptidyl-1-hydroxyglycine 1-amidating lyase (PAL), the second part of the bifunctional enzyme, will be used to understand PAM function. Roles for the linker regions connecting PHM and PAL will be explored in soluble PAM proteins with the goal of crystallization. This knowledge will clarify how the linkers affect the ability of membrane PAM to signal to cytosol and nucleus. AtT-20 cells will be used to determine whether membrane tethered PAM has improved access to ascorbate and copper, facilitating peptide amidation. Aim 2: Focusing on two amidated peptides, vasopressin and adrenomedullin, we will evaluate the ability of PAM mice to handle a high salt diet. The mechanisms underlying the behavioral changes observed in PAM mice will be explored in electrophysiological studies focused on GABAergic signaling in the amygdala. Aim 3: How regulated intramembrane proteolysis generates sfCD will be evaluated in PAM-1-expressing AtT-20 cells. Effects of splice variants, luminal domain cleavage and phosphorylation will be determined. Cell permeant versions of sfCD will be used to explore the effects of PAM on gene expression, focusing on PAM targets known to play roles in the secretory pathway (aquaporin1; secretory leukocyte proteinase inhibitor) and copper metabolism (Atox1). While genetic alterations in PAM may be rare, our data strongly suggest that alterations in the availability of dietary copper and ascorbate could contribute to less than adequate functioning of PAM. It is our hope that a better understanding of the copper-reversible changes that occur in PAM mice, and are mimicked in copper deficient wildtype mice, will facilitate identification of compromised PAM function in man.

描述（由申请人提供）：肽在整个内分泌和神经系统中发挥着重要作用。从前激素原到产物肽的生物合成途径在人类和海葵等原始生物中基本相似。当前体穿过分泌途径的内腔时，内切蛋白酶、外肽酶和肽酰甘氨酸 1-酰胺化单加氧酶 (PAM)（负责关键 C 末端酰胺化的酶）依次发挥作用。肽基甘氨酸 1-羟基化单加氧酶 (PHM; EC1.14.17.3) 是双功能 PAM 蛋白的第一个酶，需要铜和抗坏血酸；对于人类来说，两者都必须从饮食中获得。像PHM这样的铜酶在厌氧物种中很少见，随着大气中分子氧的出现而进化，并且与它的使用密切相关。我们对具有一种功能性 PAM 基因的小鼠（PAM 小鼠）的分析揭示了多种生理和行为改变，测得的酰胺化肽略有减少。许多改变通过额外的膳食铜得到改善，并在铜缺乏的野生型小鼠中进行模拟，从而得出 PAM 在铜稳态中发挥作用的结论。我们将基于我们的发现，即膜 PAM 产生其胞质结构域 (sfCD) 的可溶性片段，该片段靶向细胞核并改变基因表达，以确定 PAM 影响生理功能的机制。目标 1：确定的 PHM 和肽基-1-羟基甘氨酸 1-酰胺化裂解酶 (PAL)（双功能酶的第二部分）的结构将用于了解 PAM 功能。将在可溶性 PAM 蛋白中探索连接 PHM 和 PAL 的连接区域的作用，以实现结晶。这些知识将阐明连接子如何影响膜 PAM 向细胞质和细胞核发出信号的能力。 AtT-20 细胞将用于确定膜束缚的 PAM 是否改善了抗坏血酸和铜的获取，从而促进肽酰胺化。目标 2：我们将重点关注两种酰胺化肽：加压素和肾上腺髓质素，评估 PAM 小鼠处理高盐饮食的能力。 PAM 小鼠中观察到的行为变化背后的机制将在侧重于杏仁核 GABA 信号传导的电生理学研究中进行探索。目标 3：将在表达 PAM-1 的 AtT-20 细胞中评估受调节的膜内蛋白水解如何产生 sfCD。将确定剪接变体、管腔结构域切割和磷酸化的影响。 sfCD 的细胞渗透版本将用于探索 PAM 对基因表达的影响，重点关注已知在分泌途径（水通道蛋白1；分泌性白细胞蛋白酶抑制剂）和铜代谢（Atox1）中发挥作用的 PAM 靶标。虽然 PAM 的基因改变可能很少见，但我们的数据强烈表明，膳食铜和抗坏血酸可用性的改变可能导致 PAM 功能不足。我们希望更好地了解 PAM 小鼠中发生的铜可逆变化，并在铜缺乏的野生型小鼠中进行模拟，将有助于识别人类受损的 PAM 功能。