A glutathione S-transferase π-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins
A glutathione S-transferase π-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins
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DOI：
10.1124/mol.105.018523
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发表时间：
2006-02-01
影响因子：
3.6
通讯作者：
Tew, KD
中科院分区：
文献类型：
--
作者：
Townsend, DM;Findlay, VJ;Tew, KD
一氧化氮（NO）是一种内源性的、可扩散的跨细胞信使，可以影响细胞存活的调控和信号通路。暴露于NO可以对靶蛋白进行直接的翻译后修饰，如硝化和/或亚硝基化。另一种选择是，在与氧、超氧化物、谷胱甘肽或某些金属相互作用后，NO可导致s -谷胱甘肽化，这是一种翻译后修饰，对信号通路可能至关重要。一种新的谷胱甘肽s -转移酶（GST pi）激活的前药O-2-{2,4-二硝基-5[4-（N-二甲氨基）苯甲酰氧基]苯基}1-（N，N-二甲氨基）重氮-1-ium-1,2-二酸酯（PABA/NO），在体外和体内释放NO并引起毒性。我们现在表明，PABA/NO诱导亚硝化应激，导致无法检测到的亚硝基化，有限的硝化和高水平的s -谷胱甘肽化。经过单次药理学相关剂量的PABA/NO后，s -谷胱甘肽酰化迅速发生（< 5分钟），持续时间约为7小时，这意味着去谷胱甘肽酰化过程的半衰期约为3小时。二维sds -聚丙烯酰胺凝胶电泳和s -谷胱甘肽酰化残基单克隆抗体免疫印迹表明，许多蛋白质被s -谷胱甘肽酰化。随后的基质辅助激光解吸电离/飞行时间分析鉴定了10种蛋白质，包括β -乳酸脱氢酶、Rho GDP解离抑制剂β、ATP合成酶、延伸因子2、蛋白二硫异构酶、核磷蛋白-1、伴蛋白、肌动蛋白、蛋白酪氨酸磷酸酶1B （PTP1B）和葡萄糖苷酶II。此外，我们发现持续的s -谷胱甘肽化与药物诱导的应激激酶的激活暂时同步，已知应激激酶与细胞死亡途径有关。这与PABA/NO诱导s -谷胱甘肽化和PTP1B失活的事实是一致的，PTP1B是一种参与激酶失活的磷酸酶。这些影响与细胞内PABA/NO或代谢物的存在是一致的，因为过表达MRP1的细胞对药物的敏感性较低，s -谷胱甘肽化蛋白的水平降低。
Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GST pi)-activated pro-drug, O-2-{2,4-dinitro-5[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (< 5 min) and is sustained for similar to 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.