Biochemistry and Physiology of Peptide Amidation

肽酰胺化的生物化学和生理学

• 批准号: 8068433
• 负责人: ELIZABETH ANNE EIPPER
• 金额: $ 9.82万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-07 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068433
• 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; 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. PUBLIC HEALTH RELEVANCE: Bioactive peptides are among the most ancient methods for intercellular communication. Peptide amidation, the modification of the ionizable COOH-terminal of a peptide to have an uncharged amide group, requires a number of cofactors and presents a set of challenges to cells using amidated peptides for intercellular signaling, performed by bifunctional peptidylglycine alpha- amidating monooxygenase, PAM. Both enzymatic activities of PAM have been extensively studied and their enzymology and biology, along with the biological consequences of inadequate ability to perform peptide amidation, form the basis of this work.

描述(由申请人提供)：多肽在整个内分泌和神经系统中扮演着重要的角色。从激素前体到产物肽的生物合成途径在人类和原始生物(如海葵)中基本上是相似的。当前体通过分泌途径的管腔时，内切酶、外肽酶和负责关键C-末端酰胺化的肽甘氨酸1-酰胺化单加氧酶(PAM)依次发挥作用。氨基甘氨酸-1-羟基单加氧酶(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；对人类来说，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，并与其使用压倒性地相关。我们对具有一个功能PAM基因的小鼠(PAM小鼠)的分析显示出多种生理和行为变化，所测量的酰胺化多肽略有减少。许多变化可以通过添加饲料中的铜来改善，并在铜缺乏的野生型小鼠中模拟，从而得出结论，PAM在铜的动态平衡中发挥作用。我们将基于我们的发现，即膜PAM产生其胞浆结构域(SfCD)的可溶片段，该片段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。目的：通过对PHM和双功能酶的第二部分--1-羟基甘氨酸-1-氨基裂解酶(PAL)结构的测定，了解PAM的功能。将探索连接PHM和PAL的连接区在可溶性PAM蛋白中的作用，目标是结晶。这一知识将阐明连接子如何影响膜PAM向胞浆和细胞核发出信号的能力。ATT-20细胞将被用来确定膜拴系PAM是否改善了抗坏血酸和铜的获取，从而促进了多肽的酰胺化。目的2：针对加压素和肾上腺髓质素这两种酰胺化多肽，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专注于杏仁核GABA能信号的电生理学研究中进行探索。目的3：在表达PAM-1的AtT-20细胞中，将评估受调控的膜内蛋白分解如何产生sfCD。剪接变异体、腔结构域切割和磷酸化的影响将被确定。细胞水平的sfCD将被用来探索PAM对基因表达的影响，重点是已知的在分泌途径(水通道蛋白1；分泌性白细胞蛋白酶抑制物)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，饮食中铜和抗坏血酸可获得性的改变可能导致PAM功能不足。我们希望，更好地了解PAM小鼠体内发生的铜可逆变化，以及在铜缺乏的野生型小鼠中模拟的铜可逆变化，将有助于识别人类PAM功能受损。 公共卫生相关性：生物活性多肽是最古老的细胞间通讯方法之一。多肽酰胺化是将多肽的可电离的COOH末端修饰成一个不带电荷的酰胺基团，这需要许多辅因子，并对细胞提出了一系列挑战，使用酰胺化多肽进行细胞间信号转导，由双功能肽甘氨酸α-酰胺化单加氧酶(PAM)执行。PAM的两种酶活性都得到了广泛的研究，它们的酶学和生物学，以及执行肽酰胺化能力不足的生物学后果，构成了这项工作的基础。