NADK

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Introduction

Highly proliferating cancer cells require sufficient amounts of NADH and NADPH as reducing agents for the reductive synthesis of proteins, nucleic acids, and lipids (Figure 1A). The lack of these substances will cause cell growth to stop and eventually lead to cell death. NADPH also plays an important role in maintaining a healthy redox state in cells, as it is involved in neutralizing reactive oxygen species (ROS) associated with rapid growth.

Figure 1. The role of NAD⁺ kinase (NADK) and dehydrogenases (G6PD) and the malic enzymes, M1 and M2, in generating NADPH (Tedeschi, P.M.; et al. 2016.)

The reduction in NADPH levels is expected to have two effects on tumor survival: inhibit key biosynthetic pathways and reduce the ability of cancer cells to neutralize high ROS levels. There is a key role in the process of NADPH regulation: NAD⁺ kinase (NADK), which produces NADP that is then converted into NADPH under the action of dehydrogenase. NADK was isolated and identified from Saccharomyces cerevisiae as early as 1950, and has since been purified from a variety of organisms.

Human cytoplasmic NADK (cNADK) is an obligate dimer with a monomer size of approximately 50 kDa. Human cNADK cannot use inorganic phosphate. The active site of the enzyme is composed of portions of two monomers, which is a distinctive feature of the enzyme (Figure 1B). The structure and function of human cNADK have been known for more than ten years. Recently, it has been discovered that mitochondrial NADK (mNADK) may play an important role in controlling the ROS produced in mitochondria. So far, due to the differences in the structure and substrate between prokaryotic and mammalian NADK active sites, most of the efforts to study NADK inhibitors have focused on pathogenic bacteria. At present, a lot of efforts have been made to research NADK inhibitors for multidrug-resistant malaria and tuberculosis. Inhibition of human NADK for the treatment of diseases such as inflammation and cancer has not been proposed until recently.

Figure 2. Model of NAD⁺ (space-filling model) bound to human NADK with each monomer colored green and cyan created using PyMOL (Tedeschi, P.M.; et al. 2016.)

Mitochondrial-localizated of NADK

Although NADPH and NADK play a vital role in regulating cellular ROS, recent studies have shown that the knockdown of cNADK has only a modest effect on ROS levels. Other studies have shown that cNADK (cytoplasmic NADK) only leads to minimal protection of ROS. Pollak and colleagues believe that human cells may rely more on NADP⁺-dependent dehydrogenase to reduce NADP⁺ than NADP⁺ levels for oxidative defense. The recently identified mitochondrial NADK (mNADK) can also be used to explain this result. Since NADP is impermeable across the membrane, it has long been hypothesized that mitochondrial NADK exists, and it has not been characterized until recently. Mitochondria NADH and NADPH are necessary for protection from oxidative stress and macromolecular biosynthesis. mNADK may play an indispensable role in the production of NADH and NADPH to protect mitochondria from ROS. Due to the increased levels of ROS in tumor cells, mNADK inhibition seems to be a type of strategy for cancer treatment. However, since this enzyme may play a key role in protecting the heart and brain against ROS, this method may be too toxic for clinical use and must undergo a lot of careful testing.

Cytoplasmic NADK expression in cancer

A new cNADK mutant NADK-I90F was isolated from the mutants in pancreatic ductal adenocarcinoma cancer patients, and its molecular structure and enzyme activity were subsequently characterized. Compared with the wild-type enzyme, cNADK-I90F cNADK-I90F has a higher Vmax and lower Km for NAD⁺ and ATP, indicating that its activity is increased. Consistent with this finding, cells expressing this mutant have elevated NADPH levels and low ROS levels. When studying the effects of NADK-I90F on cells in vitro and in vivo, it was found that its increased enzyme activity allows it to drive the transformation of normal pancreatic duct cells. This further supports the view that wild-type and certain mutant forms of cNADK are clinically relevant enzyme targets for cancer therapy.