An NADPH Sensor Protein (HSCARG) Down-regulates Nitric Oxide Synthesis by Association with Argininosuccinate Synthetase and Is Essential for Epithelial Cell Viability

Abstract

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.Nitric oxide (NO), 2 a cellular signaling molecule, has been shown to be involved in vascular regulation, autoimmunity, and neurotransmission and impacts diverse biological processes, including cell survival (1-7). The impaired production of NO can result in the vascular dysfunction, whereas overproduction of NO will induce some diseases such as the cerebral infarction, diabetes mellitus, and neurodegenerative disorders (7-11). Arginine is the sole amino acid substrate that is required for the production of NO (12, 13), and its regeneration from citrulline, the co-product of NO synthesis, is rate-limited by argininosuccinate synthetase (AS) (13-16). In addition, the reducing reagent donor, NADPH, and oxygen are necessary for NO production (13, 17). For this reason, NO production is not only limited by the regeneration of arginine but is also affected by the intracellular NADPH concentration, which requires cross-talk between the signaling pathways of NADPH and NO.In addition to its well known function in energy metabolism, NADH, along with its phosphorylated relative NADPH, has been recognized as an important regulatory molecule. Together, their roles are crucial in signaling pathways that control fundamental cellular processes, such as transcription, regulation of calcium homeostasis, and apoptosis (18 -20). NAD mainly exists in its oxidized state (NAD ϩ ), whereas NADP is largely found in its reduced form, NADPH (21, 22). The predominant function of NADP is to maintain a pool in its reduced form to ensure a rapid regeneration of the defense systems to protect cells from oxidative damage. NADPH holds a key position in...

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“…The negative effect of HSCARG on NO production is enhanced when the intracellular NADPH/NADP ϩ ratio decreases. In response to DHEA or SNAP treatment, HSCARG translocates from the cytoplasm to the nucleus (28,29), suggesting that HSCARG may exert regulatory functions in the nucleus as well.…”

“…In our previous work, we have solved the crystal structure (29) and found that it is involved in regulation of NO production (28). HSCARG interacts with argininosuccinate synthetase, a rate-limiting enzyme in NO synthesis, and down-regulates NO synthesis through repression of argininosuccinate synthetase activity.…”

“…HSCARG is a NADPH sensor, which plays a regulatory role in cellular processes (28,29). In our previous work, we have solved the crystal structure (29) and found that it is involved in regulation of NO production (28).…”