Mitochondrial NAD Kinase, a Key Player in NADP(H) Metabolism and its Implications in Health, Disease and Therapeutics

NADPH provides the necessary reducing power for various biosynthetic reactions, such as fatty acid synthesis. Additionally, NADPH is crucial for detoxifying reactive oxygen species in cells, helping to protect the cell from oxidative damage. This critical coenzyme exists in two forms: oxidized (NADP⁺) and reduced (NADPH), collectively referred to as NADP or NADP(H). NADP(H) is essential in redox reactions, wherein it serves as a source of electrons, particularly in defending against oxidative stress by replenishing antioxidants like glutathione and thioredoxin to neutralize reactive oxygen species (ROS). The enzyme NAD kinase (NADK) is responsible for phosphorylating NAD to generate NADP, and its indispensable nature is reflected in its presence across all life kingdoms. However, subcellular compartmentalization necessitates distinct NADKs in eukaryotic cells, with specific NADKs responsible for maintaining NADP levels in separate compartments. Notably, mitochondrial NADP holds particular significance due to the mitochondria’s role as a major source of ROS production and its critical involvement in energy metabolism. While bacteria possess a single NADK for NADP synthesis, eukaryotic cells require compartment-specific NADKs for both the cytosol and mitochondria. For instance, in yeast, Pos5 encodes mitochondrial NADK, while Utr1 and Yef1 encode cytosolic NADKs. In the model plant Arabidopsis thaliana, distinct NADKs are localized to the cytosol, chloroplasts, and peroxisomes. The human NADK, however, was initially identified solely in the cytosol, leaving the identity of the mitochondrial NADK a mystery for over a decade.

In a new paper published in Redox Biology, an official Journal of the Society for Redox Biology and Medicine, by Drs. Ren Zhang and Kezhong Zhang from Wayne State University in Detroit, they explored the significance of NADK2 in mitochondrial function, its role in human health and disease, and the potential for NADK2 as a therapeutic target. According to the authors, the discovery of NADK2, also known as MNADK, marked a significant breakthrough in our understanding of NADP metabolism within the mitochondria. Dr. Kousaku Murata’s group performed a BLASTP search using atNADK3, an Arabidopsis thaliana mitochondrial NADK, which led to the unexpected detection of its human homolog, C5ORF33, in the human genome. Subsequent experiments using yeast complementation demonstrated that C5ORF33 could rescue the lethality resulting from the deletion of three yeast NADKs (Utr1, Yef1, and Pos5), indicating its ability to generate NADP(H). In vitro studies confirmed the NADK activity of recombinant C5ORF33, and it was revealed that C5ORF33 could phosphorylate both NAD⁺ and NADH, albeit with a lower efficiency for NADH. C5ORF33 was found to localize exclusively within the mitochondria of human cells. Meanwhile, the research group led by Dr. Ren Zhang at Wayne State University independently identified C5ORF33 as a mitochondrial NAD kinase, (hence the name MNADK) through an RNA-seq screening designed to identify nutritionally-regulated genes in the liver and fat. The results showed a significant increase in C5ORF33 expression levels in response to fasting in both the liver and white adipose tissue. His group identified a conserved NADK domain in C5ORF33, and then expressed and purified the recombinant protein. Using mass spectrometry, his group demonstrated that the recombinant C5ORF33 protein phosphorylated NAD⁺ to generate NADP⁺. Further analysis of tissue distribution revealed that C5ORF33 expression was highest in the liver but also notably high in mitochondrion-rich tissues such as brown fat, heart, kidney, muscle, and brain. Subsequent experiments, including fluorescence imaging, confirmed the mitochondrial localization in liver cells, and thus his group named the protein mitochondrial NAD kinase (MNADK), which was later officially named as NADK2 (Figure 1).

Mutations in the NADK2 gene can lead to NADK2 deficiency, a rare genetic disorder that affects mitochondrial NADP(H) metabolism. This condition can manifest with a range of symptoms, including developmental delays, intellectual disabilities, and movement disorders. Studying NADK2 deficiency cases has provided insights into the importance of this enzyme in human health. The authors presented clinical cases to demonstrate the severe metabolic and developmental challenges when NADK2 is deficient or mutated. The genetic link between the NADK2 gene and these abnormalities sheds light on its importance. By understanding the symptoms and challenges associated with NADK2 deficiencies, medical professionals can better diagnose, treat, and manage related conditions. NADK2 is also overexpressed in certain cancer types, and its upregulation is associated with increased mitochondrial NADP(H) levels, which contribute to tumor growth and survival. Understanding the role of NADK2 in cancer biology has raised interest in targeting this enzyme for cancer therapy. The authors discussed the relation of mitochondrial dysfunction and oxidative stress in neurodegenerative disorders. Modulating NADK2 activity or its downstream effects on mitochondrial NADP(H) metabolism may hold therapeutic potential in managing conditions like Alzheimer’s and Parkinson’s diseases. They also highlighted the imbalances in cellular NADP(H) levels can contribute to metabolic disturbances, such as insulin resistance and obesity. Investigating NADK2’s role in these conditions could lead to insights into new therapeutic strategies.

Mouse models have long served as invaluable tools for unraveling the complexities of human biology and disease. The authors discussed mouse models with altered NADK2 expression or function and how this can help researchers study the enzyme’s role in health and disease. Mouse models, especially the ones developed by the International Mouse Phenotyping Consortium (IMPC), serve as pivotal tools in understanding the function and significance of NADK2 in mammals. These models could be especially valuable in understanding the subtler effects of reduced NADK2 activity in specific tissues or cellular contexts. Such research may uncover novel insights into NADK2-related biology and potential therapeutic interventions. The crystal structures of human NADK2 have been solved by X-ray crystallography, and NADK2 forms a dimer with a perfect 2-fold symmetry, with NADP binding site at the dimer interface (Figure 2).

Drs. Ren Zhang and Kezhong Zhang then highlighted the potential of NADK2   as a therapeutic target. The critical role of mitochondrial NADP(H) in redox homeostasis and cellular energetics makes NADK2 an attractive candidate for intervention in various diseases, including those involving mitochondrial dysfunction, oxidative stress, and metabolic disturbances. Strategies aimed at modulating NADK2 activity could potentially impact these conditions positively. For instance, developing small molecules that selectively inhibit NADK2 activity could help manage conditions characterized by excessive mitochondrial NADP(H) levels, such as certain cancer types where NADK2 is overexpressed. Moreover, for NADK2-deficient patients, gene therapy approaches aimed at restoring functional NADK2 expression in mitochondria hold great promise. Advances in gene editing technologies, such as CRISPR-Cas9, offer exciting opportunities for precisely correcting NADK2 mutations. Considering the potential for NADH supplementation in managing NADK2 deficiency, further exploration of metabolite-based therapies is warranted. These therapies may involve targeted supplementation of NAD(P)(H) precursors or cofactors to improve mitochondrial redox balance. Indeed, understanding the role of NADK2 in specific diseases, such as neurodegenerative disorders or mitochondrial diseases, may lead to tailored therapeutic strategies. Targeting NADK2-related pathways in disease-specific contexts could yield effective treatments.

In essence, the expert opinion and comprehensive review of Drs. Ren Zhang and Kezhong Zhang enriches our understanding of NADP metabolism, emphasizing the importance of NADK2 in maintaining cellular health. The knowledge can pave the way for new therapeutic strategies targeting a range of diseases.