Biochemistry and Physiology of Peptide Amidation

肽酰胺化的生物化学和生理学

• 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
• 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

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扮演重要角色。在人类和原始生物（例如海葵）中，从前激素到产物肽的生物合成途径从根本上相似。随着前体穿过分泌途径的腔，内螺旋蛋白酶，外肽酶和肽基甘氨酸1-增强单氧酶（PAM），酶是负责至关重要的C-末端酰化的酶。肽基甘氨酸1-羟基化单加氧酶（PHM; EC1.14.17.3）是双功能PAM蛋白的第一种酶，需要铜和抗坏血酸；在人中，两者都必须从饮食中获取。 PHM等杯状酶在厌氧物种中很少见，随着大气中的分子氧的出现而演变，并且与其使用极为相关。我们用一种功能性PAM基因（PAM小鼠）对小鼠进行的分析表明，测得的胺化肽的多次生理和行为改变，略有降低。许多改变通过额外的饮食铜来改善，并模仿铜缺乏的野生型小鼠，得出的结论是，PAM在铜稳态中起着作用。我们将基于发现膜PAM产生其胞质结构域（SFCD）的可溶片段，该碎片针对细胞核并改变基因表达，以确定PAM影响生理功能的机制。 AIM 1：针对PHM和肽基-1-羟基甘氨酸1-增强裂解酶（PAL）确定的结构（双功能酶的第二部分）将用于了解PAM功能。连接PHM和PAL的接头区域的角色将在可溶性PAM蛋白中探索，其目的是结晶。这些知识将阐明接头如何影响膜PAM信号向细胞质和核发信的能力。 ATT-20细胞将用于确定膜束缚的PAM是否可以改善抗坏血酸和铜的机会，从而促进肽胺。 AIM 2：专注于两种胺化肽，加压素和肾上腺肾上腺素，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基础机制将在杏仁核中的GABA能信号转导的电生理研究中探索。 AIM 3：将如何在表达PAM-1的ATT-20细胞中评估受调节的膜内蛋白水解产生SFCD。将确定剪接变体，腔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，重点是已知在分泌途径中起作用的PAM靶标（Aquaporin1；分泌白细胞蛋白酶蛋白酶抑制剂）和铜代谢（ATOX1）。尽管PAM的遗传改变可能很少见，但我们的数据强烈表明，饮食铜和抗坏血酸的可用性改变可能导致PAM功能不足。我们希望对PAM小鼠中发生的铜可逆变化有更好的了解，并模仿缺乏铜的野生型小鼠，将有助于识别人类的PAM功能。