Biochemistry and Physiology of Peptide Amidation

肽酰胺化的生物化学和生理学

• 批准号: 8622188
• 负责人: ELIZABETH ANNE EIPPER
• 金额: $ 54.87万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2015-07-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622188
• 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

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重要作用。从激素原到肽产物的生物合成途径在人类和原始生物如海葵中基本相似。随着前体移动通过分泌途径的腔，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）依次起作用。肽基甘氨酸1-羟基化单加氧酶（PHM; EC1.14.17.3），双功能PAM蛋白的第一种酶，需要铜和抗坏血酸;在人，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，并且与其使用密切相关。我们对具有一个功能性PAM基因的小鼠（PAM小鼠）的分析揭示了多种生理和行为改变，其中所测量的酰胺化肽略有减少。许多改变改善了额外的饮食铜和铜缺乏的野生型小鼠中模仿，导致的结论是PAM在铜稳态中发挥作用。我们将建立在我们的发现，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：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，确定的结构，将用于了解PAM功能。连接PHM和PAL的接头区域的作用将在可溶性PAM蛋白中进行探索，目的是结晶。这些知识将阐明接头如何影响膜PAM向胞质溶胶和细胞核发出信号的能力。AtT-20细胞将用于确定膜系留的PAM是否具有改善的抗坏血酸盐和铜的接近性，从而促进肽酰胺化。目的2：聚焦于两种酰胺化肽，加压素和肾上腺髓质素，我们将评估PAM小鼠处理高盐饮食的能力。在PAM小鼠中观察到的行为变化的潜在机制将在集中于杏仁核中的GABA能信号传导的电生理学研究中探索。目的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（Atox 1）中发挥作用的PAM靶标。虽然PAM的遗传改变可能是罕见的，但我们的数据强烈表明，膳食铜和抗坏血酸盐的可用性的改变可能导致PAM的功能不足。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，将有助于鉴定人体中受损的PAM功能。