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The redox regulation of melatonin synthesis in circadian rhythm

昼夜节律中褪黑激素合成的氧化还原调节

基本信息

批准号：
16590049
负责人：
TSUBOI Seiji
金额：
$ 2.3万
依托单位：
SHUJITSU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590049/
关键词：
melatonin serotonin N-acetyltransferase redox regulation circadian rhythm disulfide bonde glutathione pinal gland

项目摘要

Serotonin N-acetyltransferase (EC.2.3.1.87)(AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant-AA-NAT activity was irreversibly inhibited by N-ethylmaleimide(NEM) in an acetyl CoA-protectable manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl CoA-protectable manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [^<14>C]-NEM. Cys61 and Cys177 were specifically labeled in an acetyl CoA-protectable manner. The AA-NAT with the Cys61 to Ala and Cys177 to Ala double substitutions(Cys61Ala/Cys177Ala-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, while the AA-NATs with only the single substitutions retained about 40 % of these sensitivities. An intramolecular disulfide bond between Cys61 and Cys177 formed upon oxidation and cleaved upon reduction was identified. Furthermore, Cys61Ala/Cys177Ala-AA-NAT expressed in COS7 cells was relatively insensitive to H2O2-evoked oxidative stress, while wild type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys61 and Cys177 produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.

5-羟色胺N-乙酰转移酶(EC.2.3.1.87)(AA-NAT)是松果体中一种褪黑素节律产生酶。为了建立褪黑激素的节律，AA-NAT的活性通过几个信号通路进行精确的调节。在这里，我们展示了AA-NAT活性的新调节，其中分子内的二硫键可能作为催化的开关。N-乙基马来酰亚胺(NEM)以乙酰辅酶A保护的方式不可逆地抑制重组AA-NAT的活性。氧化的谷胱甘肽或溶解氧以乙酰辅酶A保护的方式可逆地抑制AA-NAT。为了确定与抑制有关的半胱氨酸残基，首先用溶氧氧化AA-NAT，用NEM处理，用二硫苏糖醇还原，然后用[^&lt；14&gt；C]-NEM标记。Cys61和Cys177以乙酰辅酶A保护的方式被特异性标记。具有Cys61到Ala和Cys177到Ala双取代的AA-NAT(Cys61Ala/Cys177Ala-AA-NAT)是完全活性的，但对氧化和NEM都不敏感，而只有单一取代的AA-NAT保留了约40%的敏感性。确定了Cys61和Cys177之间在氧化时形成的分子内二硫键，并在还原时断裂。此外，在COS7细胞中表达的Cys61Ala/Cys177Ala-AA-NAT对H_2O_2诱导的氧化应激相对不敏感，而野生型AA-NAT在相同条件下被强烈抑制。这些结果表明，Cys61和Cys177之间二硫键的形成和断裂产生了AA-NAT的活性状态和非活性状态。细胞内的氧化还原条件可能通过分子内二硫键的开关来调节AA-NAT的活性。