Isolation of activating factors of serotonin N-acetyltransferase from rice peptides
Isolation of activating factors of serotonin N-acetyltransferase from rice peptides
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DOI：
10.1016/j.jff.2017.12.051
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发表时间：
2018-02
影响因子：
5.6
通讯作者：
C. Moritani;Kayoko Kawakami;A. Fujita;K. Kawakami;H. Shimoda;T. Hatanaka;S. Tsuboi
中科院分区：
文献类型：
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作者：
C. Moritani;Kayoko Kawakami;A. Fujita;K. Kawakami;H. Shimoda;T. Hatanaka;S. Tsuboi
5-羟色胺N-乙酰基转移酶(AA-NAT)是褪黑素合成的限速酶，它将5-羟色胺转化为5-乙酰基5-羟色胺。我们以前报道过，细胞内的氧化还原条件通过以谷胱甘肽(GSH)依赖的方式切换分子内二硫键来调节AA-NAT的活性；因此，增加GSH水平可以激活AA-NAT。在这里，我们发现商用大米肽增加了细胞内GSH水平，并减弱了过氧化氢诱导的表达AA-NAT的COS7细胞中AA-NAT活性的失活。用Sep-Pak C18色谱柱和三轮反相高效液相色谱柱纯化多肽，然后用质谱仪进行序列分析，从而鉴定了三种多肽。其中两个肽(Pep2，VVTFGPSGLTTEVK；Pep3，YQQQFQQFLPEGQSQSQK)可防止过氧化氢对AA-NAT活性的失活。只有Pep3提高了细胞内GSH水平，并阻止了过氧化氢引起的还原/氧化GSH比率的降低。这些结果表明，Pep3通过增加细胞内GSH水平来激活AA-NAT，从而切换AA-NAT的分子内二硫键。
SerotoninN-acetyltransferase (AA-NAT), which converts serotonin toN-acetylserotonin, is a rate-limiting enzyme for melatonin synthesis. We previously reported that intracellular redox conditions regulate AA-NAT activity by switching an intramolecular disulfide bridge in a glutathione (GSH)-dependent manner; thus, increased GSH levels can activate AA-NAT. Here, we found that commercially available rice peptides increased intracellular GSH levels and attenuated H2O2-induced inactivation of AA-NAT activity in COS7 cells expressing AA-NAT constitutively. Purification of the peptides using a Sep-Pak C18 cartridge and three rounds of reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of three peptides. Two of these peptides (Pep2, VVTFGPSGLTTEVK; Pep3, YQQQFQQFLPEGQSQSQK) prevented inactivation of AA-NAT activity by H2O2. Only Pep3 increased intracellular GSH levels and prevented the decrease in the reduced/oxidised GSH ratio induced by H2O2. These results suggest that Pep3 activates AA-NAT by increasing intracellular GSH levels, which switches the intramolecular disulfide bond of AA-NAT.