image: (A) Tetrameric organization of human NADK. Four protomers are presented in different colors, and the N-terminal residues of each protomer are labeled. (B) Close-up view of NADK monomer. The N-terminal region (grey), N-terminal domain (NTD; green), C-terminal domain (CTD; cyan), connecting β strands 6 and 19 (magenta and orange), C-terminal α helix (blue), and three glycine-rich motifs (red) in a NADK monomer are color-coded, and secondary structural modules are labeled. (C, D) Two different views of NADK subdomains in the tetramer.
Credit: Genes & Diseases
NAD(H) and NADP(H) are important electron carriers in cellular metabolism that also act as essential cofactors for a range of enzymes involved in various biological processes. Nicotinamide adenine dinucleotide (NAD+) kinase (NADK), which catalyzes the phosphorylation of NAD+ to NADP+, is a key regulator of cellular NAD+/NADP+ homeostasis.
Alterations in the protein expression or enzymatic activity of NADK disturb cellular NAD+/NADP+ levels, leading to enhanced ROS levels that may promote cancer progression and metastasis. Previous studies have shown that mutations in cytoplasmic NADK (cNADK) are associated with increased NADPH levels and reduced ROS levels, highlighting the important role of the N-terminal region of cNADK in regulating its function.
A recent study published in the Genes & Diseases journal by researchers from Renmin Hospital of Wuhan University, the Chinese University of Hong Kong (Shenzhen), the Medical School of Xi'an Jiaotong University and Georgetown University Medical Center investigates the regulatory mechanism of cNADK and characterizes the effects of cancer-associated NADK mutants on NADK activity and cancer cell response to chemotherapy.
The authors conducted a cryo-EM single-particle study on a full-length NADK protein to understand the structural basis of enzymatic catalysis and its regulatory mechanisms. The tetrameric structure of NADK revealed a dimer-of-dimers arrangement. The catalytic domain of NADK comprises an N-terminal domain (NTD) and a C-terminal domain (CTD). A C-terminal αC helix from a neighboring monomer situated at the dimer interface aids CTD-mediated NADK tetramerization.
A comparison of the X-ray crystallography and cryo-EM structures of the human NADK tetramer shows a major difference in the interaction of the N-terminal region within the dimers, which regulates NTD orientation and also affects its substrate binding and catalytic activity. This conformational change occurs within the NADK dimer, which then forms a tetramer (a dimer of dimers). Further experiments with purified WT, C349S, R356A and Y327A mutant NADK proteins showed that a stable NADK tetramer conformation enhances substrate binding and subsequent enzyme activity, while mutations impacting the tetramer formation may inhibit its activity.
The authors further showed that R45H mutation (in the N-terminal region) enhances NADK activity, while methylation of NADK R45 attenuates it. In vivo studies revealed that the NADK R45H mutant generates more NADP+ and NADPH, leading to decreased ROS levels, enhanced tumor growth, and chemotherapy resistance. Mutations at the tetramer interface, like Ala330 (A330V mutant) and Asp314 (D314H mutant) were shown to suppress NADK activity. These loss-of-function NADK mutants reduced NADP/NADPH production, leading to increased ROS levels that inhibit cancer cell growth, proliferation, and colony formation, while increasing sensitivity to chemotherapy.
In conclusion, the findings of this study provide insights into the mechanisms regulating NADK activity and identified various mutant NADK phenotypes that modulate NADK activity, resulting in either enhanced tumor progression and chemotherapy resistance or suppression of tumor growth with increased chemotherapy sensitization.
Reference
Title of the original paper: NADK tetramer defective mutants affect lung cancer response to chemotherapy via controlling NADK activity
Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.
DOI: https://doi.org/10.1016/j.gendis.2024.101510
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