Pollak Nadine, Niere Marc, Ziegler Mathias
Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5008 Bergen, Norway.
Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5008 Bergen, Norway.
J Biol Chem. 2007 Nov 16;282(46):33562-33571. doi: 10.1074/jbc.M704442200. Epub 2007 Sep 13.
NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H(2)O(2). Overexpression of NADK resulted in a 4-5-fold increase in the NADPH, but not NADP(+), concentration, although the recombinant enzyme phosphorylated preferentially NAD(+). Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP(+)), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress.
NAD激酶（NADKs）至关重要，因为它们能生成细胞内的NADP库。与植物和酵母中的三种特定亚细胞定位的同工型不同，在哺乳动物中仅鉴定出一种NADK，我们通过免疫细胞化学确定了其胞质定位。为了解人类该酶的生理作用，我们构建并分析了稳定缺失或过表达NADK的细胞系。短发夹RNA介导的下调导致NADK表达、活性和NADPH浓度均出现相似的（约70%）下降，并伴随着对H₂O₂敏感性的增加。NADK的过表达导致NADPH浓度增加4至5倍，但NADP⁺浓度未增加，尽管重组酶优先磷酸化NAD⁺。令人惊讶的是，NADK过表达以及随之而来的NADPH水平升高仅适度增强了对氧化处理的保护作用。显然，为维持氧化防御系统再生所需的NADPH水平，人类细胞主要依赖于NADP依赖性脱氢酶（其将NADP⁺重新还原），而非NADP的净增加。所构建细胞系中NADPH水平的稳定变化还伴随着过氧化物酶体增殖物激活受体5和核因子E2相关因子2表达的改变。由于细胞系中的基础氧自由基水平仅略有变化，NADP的氧化还原状态可能是氧化应激的主要传递者。
