Biochemistry and Physiology of Peptide Amidation

描述（由申请人提供）：多肽在整个内分泌和神经系统中发挥重要作用。从激素前体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。当前体通过分泌途径的管腔时，内源性蛋白酶、外肽酶和负责关键C端酰胺化的肽基甘氨酸1-酰胺化单加氧酶（PAM）依次发挥作用。肽基甘氨酸1-羟化单加氧酶（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。我们对具有一个功能性PAM基因的小鼠（PAM小鼠）的分析显示出多种生理和行为改变，所测量的修饰肽略有减少。许多改变通过额外的饮食铜得到改善，并在缺铜野生型小鼠中进行了模拟，从而得出PAM在铜稳态中起作用的结论。我们将建立在我们的发现，膜PAM产生一个可溶性片段的细胞质结构域（sfCD）是针对细胞核和改变基因表达，以确定通过PAM影响生理功能的机制。目的1：确定PHM和双功能酶的第二部分肽基-1-羟基甘氨酸1-酰胺裂解酶（PAL）的结构，将用于了解PAM的功能。连接PHM和PAL的连接区域在可溶性PAM蛋白中的作用将以结晶为目标进行探索。这一知识将阐明连接物如何影响膜PAM向胞浆和细胞核发出信号的能力。AtT-20细胞将用于确定膜系系PAM是否改善了抗坏血酸和铜的通路，促进了肽酰胺化。目的2：以抗利尿激素和肾上腺髓质素两种修饰肽为重点，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机制将在以杏仁核gaba能信号为重点的电生理学研究中进行探讨。目的3：将在表达pam -1的at -20细胞中评估受调控的膜内蛋白水解如何产生sfCD。剪接变异体、管腔结构域切割和磷酸化的影响将被确定。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PAM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）中发挥作用的靶点。虽然PAM的遗传改变可能是罕见的，但我们的数据强烈表明，饮食中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，并在缺铜野生型小鼠中进行模拟，将有助于识别人类受损的PAM功能。

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.