The crystal structure of the protein known as HSCARG shows that it binds NADPH (nicotinamide adenine dinucleotide phosphate), and changes in the ratio of NADPH to its oxidized form NADP+ alter the subcellular localization of HSCARG, which suggests that it may serve as a sensor for NADPH. One of the important functions of NADPH is in the production of nitric oxide (NO). The substrate for nitric oxide synthase (NOS) is arginine, which is regenerated from citrulline through a reaction involving the rate-limiting enzyme argininosuccinate synthetase (AS). Zhao et al. showed that AS was coimmunoprecipitated with HSCARG from HeLa cells, and this interaction was further characterized in transfected cells and in vitro assays. In transfected cells, the two proteins colocalized, and overexpression of AS resulted in the partial translocation of HSCARG from the cytoplasm to the nucleus. This translocation was also noted when the cells were treated with an NO donor and was inhibited in the presence of a cGMP inhibitor. The abundance of HSCARG was also increased in cells overexpressing AS, and the NO donor or inflammatory cytokines that raise NO concentrations, such as tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), also stimulated transcription of hscarg. In cells overexpressing HSCARG, NO production was decreased and HSCARG inhibited AS activity in vitro. Cells in which HSCARG was decreased through RNA interference exhibited elevated NO production and were more susceptible to apoptosis. Dehydroepiandrosterone (DHEA) was used to decrease the NADPH/NADP+ ratio and, in cells cotransfected with tagged versions of HSCARG and AS, an increase in the interaction between these two proteins was observed after treatment with DHEA. Addition of NADPH to purified HSCARG and AS inhibited the interaction of the proteins in vitro. Thus, HSCARG appears to allow cells to respond to changing redox conditions and contribute to the regulation of NO production. It will be interesting to determine its function in the nucleus.
Y. Zhao, J. Zhang, H. Li, Y. Li, J. Ren, M. Luo, X. Zheng, An NADPH sensor protein (HSCARG) down-regulates nitric oxide synthesis by association with argininosuccinate synthetase and is essential for epithelial cell viability. J. Biol. Chem. 283, 11004-11013 (2008). [Abstract] [Full Text]