O-Aminobenzoyl-S-nitrosoglutathione: A fluorogenic, cell permeable, pseudo-substrate for S-nitrosoglutathione reductase.
O-Aminobenzoyl-S-nitrosoglutathione: A fluorogenic, cell permeable, pseudo-substrate for S-nitrosoglutathione reductase.
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O-氨基苯甲酰基-S-亚硝基谷胱甘肽：一种荧光、细胞可渗透的 S-亚硝基谷胱甘肽还原酶的假底物。
DOI：
10.1016/j.freeradbiomed.2017.04.008
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发表时间：
2017
影响因子：
7.4
通讯作者：
Mutus,Bulent
中科院分区：
文献类型：
--
作者：
Sun,BeiLei;Palmer,Lisa;Alam,ShaguftaRehman;Adekoya,Itunuoluwa;Brown-Steinke,Kathleen;Periasamy,Ammasi;Mutus,Bulent
S-亚硝基谷胱甘肽还原酶（GSNOR）是一种多功能酶。它可以催化S-亚硝基谷胱甘肽（GSNO）的NADH依赖性还原;以及羟甲基谷胱甘肽（HMGSH;甲醛和谷胱甘肽之间自发反应形成的加合物）的NAD+依赖性氧化。虽然最初被认为是参与甲醛解毒的酶，但越来越多的证据表明，GSNOR还通过调节蛋白质S-亚硝基硫醇信号传导在一氧化氮介导的信号传导中发挥重要作用。在人类中，GSNOR/S-亚硝基硫醇与几种疾病的病因学有关，包括肺癌、囊性纤维化、哮喘、肺动脉高压和神经元功能障碍。目前，还无法监测GSNOR在活细胞中的活性。在这篇文章中，我们提出了一种新的化合物，O-氨基苯甲酰基-S-亚硝基谷胱甘肽（OAbz-GSNO），它作为GSNOR的荧光假底物，估计Km值为320 µM。当其S-NO部分被还原时，弱的OAbz-GSNO荧光增加约14倍。在活细胞成像研究中，OAbz-GSNO容易被原代肺内皮细胞摄取，并定位于与GSNOR相同的核周区域。核周OAbz-GSNO荧光以时间依赖性方式增加，并且这种荧光增加通过siRNA敲低GSNOR或通过用GSNOR特异性抑制剂N6022和C3处理而消除。总之，这些数据表明，OAbz-GSNO可以用作监测活细胞中GSNOR活性的工具。
S-nitrosoglutathione reductase (GSNOR) is a multifunctional enzyme. It can catalyze NADH-dependent reduction ofS-nitrosoglutathione (GSNO); as well as NAD+-dependent oxidation of hydroxymethylglutathione (HMGSH; an adduct formed by the spontaneous reaction between formaldehyde and glutathione). While initially recognized as the enzyme that is involved in formaldehyde detoxification, increasing amount of evidence has shown that GSNOR also plays a significant role in nitric oxide mediated signaling through its modulation of proteinS-nitrosothiol signaling. In humans, GSNOR/S-nitrosothiols have been implicated in the etiology of several diseases including lung cancer, cystic fibrosis, asthma, pulmonary hypertension, and neuronal dysfunction. Currently, it is not possible to monitor the activity of GSNOR in live cells. In this article, we present a new compound,O-aminobenzoyl-S-nitrosoglutathione (OAbz-GSNO), which acts as a fluorogenic pseudo-substrate for GSNOR with an estimatedKmvalue of 320 µM. The weak OAbz-GSNO fluorescence increases by approximately 14 fold upon reduction of itsS-NO moiety. In live cell imaging studies, OAbz-GSNO is readily taken up by primary pulmonary endothelial cells and localizes to the same perinuclear region as GSNOR. The perinuclear OAbz-GSNO fluorescence increases in a time dependent manner and this increase in fluorescence is abolished by siRNA knockdown of GSNOR or by treatment with GSNOR-specific inhibitors N6022 and C3. Taken together, these data demonstrate that OAbz-GSNO can be used as a tool to monitor the activity of GSNOR in live cells.