Pineal Regulation: Control of arylalkylamine N-acetyltransferase

松果体调节：芳烷基胺 N-乙酰转移酶的控制

• 批准号: 7968763
• 负责人: David Klein
• 金额: $ 57.63万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968763
• 关键词: 14-3-3 Proteins; 4' -phosphopantetheine; Active Sites; Address; Affinity; Amidophosphoribosyltransferase; Aromatic Amines; Arylalkylamine N-Acetyltransferase; Bacteria; Binding; Binding Sites; Biological; Cardiovascular system; Catalysis; Cell Culture Techniques; Cell Cycle Regulation; Cell membrane; Cells; Chordata; Chronobiology; Circadian Rhythms; Coenzyme A; Complementary DNA; Complex; Crystallography; Cyclic AMP; Cyclization; Darkness; Data; Development; Disease; Drug Kinetics; Drug Metabolic Detoxication; Elements; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Stability; Enzymes; Eukaryota; Evolution; Exhibits; Exposure to; Family; Family suidae; Fishes; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Green Algae; Grouping; Homologous Gene; Hormones; Human; In Vitro; Kinetics; Lead; Light; Lighting; Liver; Measurement; Melatonin; Messenger RNA; Metabolic Biotransformation; Modeling; Molecular; Molecular Chaperones; Molecular Weight; Movement; Output; Paracrine Communication; Pattern; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Photoreceptors; Phototransduction; Physiological; Pineal gland; Play; Process; Prodrugs; Production; Proline; Property; Proteasome Inhibitor; Protein Binding; Protein Dephosphorylation; Protein Isoforms; Proteins; Proteolysis; Psyche structure; Rattus; Regulation; Renal function; Reporting; Research; Retina; Retinal; Role; Salmo trutta; Side; Signal Transduction; Site; Sleep Disorders; Structure; Subgroup; System; Techniques; Temperature; Time; Tryptamines; Vertebrates; Work; analog; autocrine; base; cephalochordate; circadian pacemaker; enzyme activity; esterase; fungus; inhibitor/antagonist; inorganic phosphate; insight; interest; lactacystin; neurotransmission; novel; prevent; research study; suprachiasmatic nucleus; teleost fish; trafficking

AANAT as the Timezyme: "Arylalkylamine N-acetyltransferase controls daily changes in melatonin production by the pineal gland and thereby plays a unique role in biological timing in vertebrates. Arylalkylamine N-acetyltransferase is also expressed in the retina, where it may play other roles in addition to signaling, including neurotransmission and detoxification. Large changes in activity reflect cyclic 3',5'-adenosine monophosphate-dependent phosphorylation of arylalkylamine N-acetyltransferase, leading to formation of a regulatory complex with 14-3-3 proteins. This activates the enzyme and prevents proteosomal proteolysis. The conserved features of regulatory systems that control arylalkylamine N-acetyltransferase are a circadian clock and environmental lighting." From (1) Evolution of AANAT: "The melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase (AANAT) is known to have recognizable ancient homologs in bacteria and fungi, but not in other eukaryotes. Analysis of new cDNA and genomic sequences has identified several additional homologs in other groupings. First, an AANAT homolog has been found in the genome of the cephalochordate amphioxus, representing the oldest homolog in chordates. Second, two AANAT homologs have been identified in unicellular green algae. The homologs in amphioxus, unicellular green algae, fungi and bacteria are similarly primitive in that they lack sequences found in vertebrate AANATs that are involved in regulation and that facilitate binding and catalysis. In addition, all these sequences are intronless. These features are consistent with horizontal transfer of the AANAT ancestor from bacteria to green algae, fungi and chordates. Lastly, a third AANAT gene has been found in teleost fish, suggesting that AANAT genes serve multiple functions in addition to melatonin synthesis." From (2) Adaptive control of melatonin synthesis: "Pineal melatonin synthesis increases at night in all vertebrates, due to an increase in the activity of arylalkylamine N-acetyltransferase (AANAT). Melatonin is also synthesized in the retina of some vertebrates and it is generally assumed that patterns of pineal and retinal AANAT activity and melatonin production are similar, i.e. they exhibit a high-at-night pattern. However, the situation in fish is atypical because in some cases retinal melatonin increases during the day, not the night. Consistent with this, we now report that light increases the activity and abundance of the AANAT expressed in trout retina, AANAT1, at a time when the activity and abundance of pineal AANAT, AANAT2, decreases. Likewise, exposure to darkness causes retinal AANAT protein and activity to decrease coincident with increases in the pineal gland. Rhythmic changes in retinal AANAT protein and activity are 180 degrees out of phase with those of retinal AANAT1 mRNA; all appear to be driven by environmental lighting, not by a circadian oscillator. The atypical high-during-the-day pattern of retinal AANAT1 activity may reflect an evolutionary adaptation that optimizes an autocrine/paracrine signaling role of melatonin in photoadaptation and phototransduction; alternatively, it might reflect an adaptation that broadens and enhances aromatic amine detoxification in the retina.?"(From 4) Posttranslational control of AANAT via 14-3-3 interaction in the retina: "14-3-3 proteins are a ubiquitous, highly conserved family of chaperone proteins involved in signal transduction, regulation of cell cycle, intracellular trafficking/targeting, cytoskeletal structure, and transcription. Although 14-3-3 proteins are among the most abundant proteins in the CNS, very little is known about their functional roles in the vertebrate retina. In the present study, we demonstrated that photoreceptors express 14-3-3 protein(s) and identified a 14-3-3 binding partner in photoreceptor cells, the melatonin-synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Importantly, our data demonstrate that the binding of 14-3-3 to AANAT is regulated by light, with dramatic functional consequences. During the night in darkness, retinal AANAT is phosphorylated and forms a complex with 14-3-3 proteins with an apparent molecular weight of approximately 90 kDa. Phosphorylation of AANAT facilitates the binding of enzyme to 14-3-3 proteins. Within the complex, AANAT is catalytically activated and protected from dephosphorylation and degradation. Light disrupts the AANAT/14-3-3 complex, leading to catalytic inactivation, dephosphorylation, and proteolytic degradation of the enzyme. In the presence of the proteasome inhibitor, lactacystin, light results in the formation of a high molecular weight complex (>150 kDa), which may represent an intermediate in the AANAT degradation process. These findings provide new insight into the roles of 14-3-3 proteins in photoreceptor cells and to the mechanisms controlling melatonin synthesis in the vertebrate retina." (From 3) Development of an AANAT inhibitor: "Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) regulates the daily rhythm in the production of melatonin and is therefore an attractive target for pharmacologic modulation of the synthesis of this hormone. Previously prepared bisubstrate analogs show potent inhibition of AANAT but have unfavorable pharmacokinetic properties due to the presence of phosphate groups which prevents transfer across the plasma membrane. Here, we examine a bis-pivaloyloxymethylene (POM)-tryptamine-phosphopantetheine prodrug (2) and its biotransformations in vitro by homogenates and pineal cells. Compound 2 is an efficient porcine liver esterase substrate for POM cleavage in vitro although cyclization of the phosphate moiety is a potential side product. Tryptamine phosphopantetheine (3) is converted to tryptamine-coenzyme A (CoA) bisubstrate analog (1) by human phosphoribosyl pyrophosphate amidotransferase (PPAT) and dephosphocoenzyme A kinase (DPCK) in vitro. Compound 2 was found to inhibit melatonin production in rat pineal cell culture. It was also found that the POM groups are readily removed to generate 3; however, further processing to tryptamine-CoA (1) is much slower in pineal extracts or cell culture. Implications for CoA prodrug development based on the strategy used here are discussed."From (5). Differential affinities of 14-3-3 proteins for phosphorylated AANAT(pAANAT): Work in progress has revealed that all 14-3-3 proteins do not exhibit the same affinity for pAANAT. Notably, the gamma isoform appears to represent a subgroup of 14-3-3 proteins that bind two molecules of pAANAT, based on indirect binding experiments. This work is being extended in attempts to confirm this indirect indication by producing crystals of pAANAT and 14-3-3 gamma. The essential role of an AANAT proline: The available evidence indicates that AANAT is a structurally stable except for one loop, termed Loop 1, which appears to be highly mobile. In one configuration, acetylate products can easily exit the active site and in another configuration the unacetylated products are tightly bound to the enzyme. We have determined that a proline in the middle of Loop 1 plays an unpredicted role in promoting the mobility of Loop 1. It appears that this proline divides Loop 1 into two semi-independent sequences which interact with each other and prevent formation of a single stable structure, thereby enhancing the mobility of the Loop 1. This novel finding has broad significance in understanding the movement of floppy loops in other proteins. The presence of this proline is highly conserved in vertebrates and represents one of the structural changes which occured during he course of evolution of AANAT.

AANAT 作为 Timezyme：“芳烷基胺 N-乙酰转移酶控制松果体产生褪黑激素的日常变化，从而在脊椎动物的生物计时中发挥独特的作用。芳烷基胺 N-乙酰转移酶也在视网膜中表达，除了信号传导外，它还可能发挥其他作用，包括神经传递和解毒。活性的巨大变化反映了循环周期 芳基烷基胺 N-乙酰转移酶的 3',5'-单磷酸腺苷依赖性磷酸化，导致与 14-3-3 蛋白形成调节复合物。这会激活酶并防止蛋白体蛋白水解。控制芳烷基胺 N-乙酰转移酶的调节系统的保守特征是生物钟和环境 灯光。”来自 (1) AANAT 的进化：“众所周知，褪黑激素节律生成酶芳基烷基胺 N-乙酰转移酶 (AANAT) 在细菌和真菌中具有可识别的古老同源物，但在其他真核生物中却没有。对新 cDNA 和基因组序列的分析已在其他分组中发现了几个其他同源物。首先，在细菌和真菌的基因组中发现了 AANAT 同源物。 头索动物文昌鱼，代表脊索动物中最古老的同源物。其次，在单细胞绿藻中已鉴定出两种 AANAT 同源物。文昌鱼、单细胞绿藻、真菌和细菌中的同源物同样是原始的，因为它们缺乏脊椎动物AANAT中发现的参与调节并促进结合和催化的序列。此外，所有这些 序列是无内含子的。这些特征与 AANAT 祖先从细菌到绿藻、真菌和脊索动物的水平转移一致。最后，在硬骨鱼中发现了第三个 AANAT 基因，这表明 AANAT 基因除了褪黑激素合成之外还具有多种功能。”来自 (2) 褪黑激素合成的适应性控制：“由于芳基烷基胺 N-乙酰转移酶 (AANAT) 活性的增加，所有脊椎动物的松果体褪黑激素合成在夜间都会增加。褪黑激素也在一些脊椎动物的视网膜中合成，通常认为松果体和视网膜 AANAT 活性和褪黑激素产生的模式相似，即它们表现出 高夜间模式。然而，鱼类的情况并不典型，因为在某些情况下，视网膜褪黑激素在白天而不是夜间增加。与此一致的是，我们现在报道，当松果体 AANAT（AANAT2）的活性和丰度降低时，光增加了鳟鱼视网膜中表达的 AANAT（AANAT1）的活性和丰度。同样，暴露在黑暗中会导致视网膜 AANAT 蛋白和活性 减少与松果体的增加同时发生。视网膜AANAT蛋白和活性的节律变化与视网膜AANAT1 mRNA的节律变化有180度的异相；所有这些似乎都是由环境照明驱动的，而不是由昼夜节律振荡器驱动的。视网膜 AANAT1 活性的非典型白天高模式可能反映了一种优化自分泌/旁分泌信号作用的进化适应 褪黑激素在光适应和光转导中的作用；或者，它可能反映了一种适应，扩大和增强了视网膜中芳香胺的解毒作用。？”（来自 4） 通过视网膜中的 14-3-3 相互作用对 AANAT 进行翻译后控制：“14-3-3 蛋白是一个普遍存在、高度保守的伴侣蛋白家族，参与信号转导、细胞周期调节、细胞内运输/靶向、细胞骨架结构和转录。虽然 14-3-3 蛋白是中枢神经系统中最丰富的蛋白之一，但人们对它们的作用知之甚少。 在脊椎动物视网膜中的功能作用。在本研究中，我们证明光感受器表达 14-3-3 蛋白，并鉴定了光感受器细胞中的 14-3-3 结合伴侣，即褪黑激素合成酶芳基烷基胺 N-乙酰转移酶 (AANAT)。重要的是，我们的数据表明 14-3-3 与 AANAT 的结合受到调节 通过光，具有戏剧性的功能后果。在黑暗的夜晚，视网膜 AANAT 被磷酸化并与 14-3-3 蛋白质形成复合物，表观分子量约为 90 kDa。 AANAT 的磷酸化促进酶与 14-3-3 蛋白的结合。在复合物内，AANAT 被催化激活并免受去磷酸化和 退化。光破坏 AANAT/14-3-3 复合物，导致酶的催化失活、去磷酸化和蛋白水解降解。在蛋白酶体抑制剂乳胞素存在下，光会导致高分子量复合物 (>150 kDa) 的形成，该复合物可能代表 AANAT 降解过程中的中间体。这些发现提供了新的 深入了解 14-3-3 蛋白在感光细胞中的作用以及控制脊椎动物视网膜中褪黑激素合成的机制。” （从 3） AANAT 抑制剂的开发：“血清素 N-乙酰转移酶（芳基烷基胺 N-乙酰转移酶，AANAT）调节褪黑激素生成的每日节律，因此是药理学调节该激素合成的一个有吸引力的靶标。之前制备的双底物类似物显示出对 AANAT 的有效抑制作用，但具有不利的药代动力学特性 由于磷酸基团的存在阻止了跨质膜的转移。在这里，我们研究了双新戊酰氧基亚甲基 (POM)-色胺-磷酸泛酰乙胺前药 (2) 及其在匀浆和松果体细胞中的体外生物转化。化合物 2 是体外 POM 裂解的有效猪肝酯酶底物，尽管磷酸盐环化 部分是潜在的副产品。色胺磷酸泛茶氨酸 (3) 在体外被人磷酸核糖焦磷酸酰胺转移酶 (PPAT) 和去磷酸辅酶 A 激酶 (DPCK) 转化为色胺辅酶 A (CoA) 双底物类似物 (1)。发现化合物 2 可抑制大鼠松果体中褪黑激素的产生 细胞培养。还发现 POM 基团很容易被除去，生成 3；然而，在松果体提取物或细胞培养物中，进一步加工成色胺辅酶 A (1) 的速度要慢得多。讨论了基于此处使用的策略的 CoA 前药开发的影响。”来自 (5)。 14-3-3 蛋白对磷酸化 AANAT (pAANAT) 的不同亲和力：正在进行的工作表明，并非所有 14-3-3 蛋白对 pAANAT 都表现出相同的亲和力。 值得注意的是，基于间接结合实验，γ亚型似乎代表了结合两个 pAANAT 分子的 14-3-3 蛋白的亚组。 这项工作正在扩展，试图通过生产 pAANAT 和 14-3-3 gamma 晶体来确认这种间接指示。 AANAT 脯氨酸的重要作用：现有证据表明，AANAT 结构稳定，除了一个称为环 1 的环外，该环似乎具有高度移动性。 在一种配置中，乙酰化产物可以轻松地离开活性位点，而在另一种配置中，未乙酰化产物与酶紧密结合。 我们确定Loop 1中间的一个脯氨酸在促进Loop 1的移动性方面发挥着意想不到的作用。看来这个脯氨酸将Loop 1分成两个半独立的序列，它们相互作用并阻止形成单一稳定结构，从而增强了Loop 1的移动性。这一新发现对于理解其他蛋白质中软盘环的移动具有广泛的意义。 这种脯氨酸的存在在脊椎动物中高度保守，代表了 AANAT 进化过程中发生的结构变化之一。