An NADPH sensor protein (HSCARG) down-regulates nitric oxide synthesis by association with argininosuccinate synthetase and is essential for epithelial cell viability
An NADPH sensor protein (HSCARG) down-regulates nitric oxide synthesis by association with argininosuccinate synthetase and is essential for epithelial cell viability
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NADPH 传感器蛋白 (HSCARG) 通过与精氨基琥珀酸合成酶结合下调一氧化氮合成，对上皮细胞活力至关重要
DOI：
10.1074/jbc.m708697200
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发表时间：
2008-04-18
影响因子：
4.8
通讯作者：
Zheng, Xiaofeng
中科院分区：
文献类型：
--
作者：
Zhao, Yanmei;Zhang, Jinfang;Zheng, Xiaofeng
NADPH是控制细胞基本过程的许多生物合成途径中的重要辅因子。我们最近测定了HSCARG的晶体结构，功能未知，并证明了它是一种NADPH传感器，可以根据细胞内NADPH/NADP水平的变化进行重组和重新分配。在本研究中，我们鉴定了一氧化氮合成中的限速酶精氨酸琥珀酸合成酶(AS)与HSCARG相关，并进一步证明HSCARG通过下调AS活性减少NO合成，而AS过表达上调hscarg mRNA转录，提示一种负反馈机制。脱氢表雄酮诱导的NADPH/NADP(+)比值下降，增强了HSCARG与AS的相互作用，从而对AS活性和一氧化氮的产生产生了更强的抑制作用。HSCARG的二聚区，153-189位氨基酸被确定与AS发生关键的相互作用。此外，HSCARG RNA干扰处理的上皮细胞存活率显著下降，同时caspase-3活性增加，这表明细胞存活率的丧失是由于细胞凋亡。这些结果表明，HSCARG对AS活性的调节对于维持细胞内氧化还原状态和一氧化氮水平之间的平衡至关重要。
NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP(+) ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.