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J. Biol. Chem., Vol. 283, Issue 16, 11004-11013, April 18, 2008

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

Yanmei Zhao, Jinfang Zhang, Huiying Li, Yiyu Li, Jie Ren, Ming Luo^¶, and Xiaofeng Zheng¹

From the National Laboratory of Protein Engineering and Plant Genetic Engineering and Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871, China and the ^¶Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

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.

Received for publication, October 22, 2007 , and in revised form, January 24, 2008.

^* This work was supported by National Science Foundation of China Grant 30670416, International Centre for Genetic Engineering and Biotechnology Project CRP/CHN05-01, and National High Technology and Development Program of China 863 Program 2006AA02A314. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed. Tel.: 86-10-6275-5712; Fax: 86-10-6276-5913; E-mail: xiaofengz{at}pku.edu.cn.

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