RÉSUMÉ
Nicotinamide adenine dinucleotide phosphate (NADP), a co-enzyme and an electron carrier, plays crucial roles in numerous biological functions, including cellular metabolism and antioxidation. Because NADP is subcellular-membrane impermeable, eukaryotes compartmentalize NAD kinases (NADKs), the NADP biosynthetic enzymes. Mitochondria are fundamental organelles for energy production through oxidative phosphorylation. Ten years after the discovery of the mitochondrial NADK (known as MNADK or NADK2), a significant amount of knowledge has been obtained regarding its functions, mechanism of action, human biology, mouse models, crystal structures, and post-translation modifications. NADK2 phosphorylates NAD(H) to generate mitochondrial NADP(H). NADK2-deficient patients suffered from hyperlysinemia, elevated plasma C10:2-carnitine (due to the inactivity of relevant NADP-dependent enzymes), and neuronal development defects. Nadk2-deficient mice recapitulate key features of NADK2-deficient patients, including metabolic and neuronal abnormalities. Crystal structures of human NADK2 show a dimer, with the NADP+-binding site located at the dimer interface. NADK2 activity is highly regulated by post-translational modifications, including S188 phosphorylation, K76 and K304 acetylation, and C193 S-nitrosylation; mutations in each site affect NADK2 activity and function. In mice, hepatic Nadk2 functions as a major metabolic regulator upon increased energy demands by regulating sirtuin 3 activity and fatty acid oxidation. Hopefully, future research on NADK2 will not only elucidate its functional roles in health and disease but will also pave the way for novel therapeutics for both rare and common diseases, including NADK2 deficiency and metabolic syndrome.
Sujet(s)
Mitochondries , NAD , Humains , Animaux , Souris , NADP/métabolisme , Mitochondries/génétique , Mitochondries/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/composition chimique , Phosphotransferases (Alcohol Group Acceptor)/génétique , Phosphotransferases (Alcohol Group Acceptor)/métabolismeRÉSUMÉ
Nicotinamide adenine dinucleotide (NAD+) kinase (NADK) phosphorylates NAD+, thereby producing nicotinamide adenine dinucleotide phosphate (NADP). Both NADK genes and the NADP(H)-producing mechanism are evolutionarily conserved among archaea, bacteria, plants and mammals. In mammals, NADK is activated by phosphorylation and protein-protein interaction. Recent studies conducted using genetically altered models validate the essential role of NADK in cellular redox homeostasis and metabolism in multicellular organisms. Here, we describe the evolutionary conservation, molecular properties, and signaling mechanisms and discuss the pathophysiological significance of NADK.
Sujet(s)
NAD , Plantes , Animaux , NAD/métabolisme , NADP/métabolisme , Plantes/métabolisme , Transduction du signal , Mammifères/métabolismeRÉSUMÉ
Cancer-associated fibroblasts (CAFs) are considered one of the most critical stromal cells that interact with pancreatic ductal adenocarcinoma (PDAC) and promote tumor growth, metastasis, and treatment resistance. Previous studies illustrated macroautophagy/autophagy contributes to CAF activation during tumor progression. Here in our study, we found that autophagy deficiency in CAFs impedes CAF activation by inhibiting proline biosynthesis and collagen production. Furthermore, we uncovered that autophagy promotes proline biosynthesis through mitophagy-mediated regulation of NADK2 (NAD kinase 2, mitochondrial), an enzyme responsible for production of mitochondrial NADP(H). Using an orthotopic mouse model of PDAC, we found that inhibiting mitophagy by targeting PRKN (parkin RBR E3 ubiquitin protein ligase) in the stroma reduced tumor weight. Thus, inhibition of CAFs mitophagy might be an attractive strategy for stroma-focused anti-cancer intervention. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/ß-actin: actin, beta; ALDH18A1/P5CS: aldehyde dehydrogenase 18 family, member A1; ATG3: autophagy related 3; ATG5: autophagy related 5; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CAFs:cancer-associated fibroblasts; COL1A1: collagen, type I, alpha 1; DES: desmin; ECM: extracellular matrix; FABP4: fatty acid binding protein 4, adipocyte; FAP/FAPα: fibroblast activation protein; IHC: immunohistochemical staining; LAMP1: lysosomal-associated membrane protein 1; NADK2: NAD kinase 2, mitochondrial; PC1: pro-collagen 1; PDAC: pancreatic ductal adenocarcinoma; PDGFR: platelet derived growth factor receptor; PDPN: podoplanin; PRKN: parkin RBR E3 ubiquitin protein ligase; PSCs: pancreatic stellate cells; VIM: vimentin; WT: wild-type.
Sujet(s)
Fibroblastes associés au cancer , Carcinome du canal pancréatique , Tumeurs du pancréas , Souris , Animaux , Autophagie , Fibroblastes associés au cancer/métabolisme , Fibroblastes associés au cancer/anatomopathologie , Actines , Tumeurs du pancréas/anatomopathologie , Carcinome du canal pancréatique/anatomopathologie , Ubiquitin-protein ligases/métabolisme , Proline , Tumeurs du pancréasRÉSUMÉ
Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8 h light / 16 h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2-, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities.
Sujet(s)
Protéines d'Arabidopsis , Arabidopsis , Arabidopsis/physiologie , Espèces réactives de l'oxygène , Peroxyde d'hydrogène , Chloroplastes/métabolisme , Protéines d'Arabidopsis/génétique , Protéines d'Arabidopsis/métabolisme , Photosynthèse/physiologieRÉSUMÉ
OBJECTIVE: The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (MNADK) mediates de novo mitochondrial NADP biosynthesis by catalyzing the phosphorylation of NAD to yield NADP. In this study, we investigated the function and mechanistic basis by which MNADK regulates metabolic homeostasis. METHODS: Generalized gene set analysis by aggregating human patient genomic databases, metabolic studies with genetically engineered animal models, mitochondrial bioenergetic analysis, as well as gain- and loss- of-function studies were performed to address the functions and mechanistic basis by which MNADK regulates energy metabolism and redox state associated with metabolic disease. RESULTS: Human MNADK common gene variants or decreased expression of the gene are significantly associated with the occurrence of type-2 diabetes, non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma (HCC). Ablation of the MNADK gene in mice led to decreased fat oxidation, coincident with increased respiratory exchange ratio (RER) and decreased energy expenditure upon energy demand triggered by endurance exercise or fasting. On an atherogenic high-fat diet (HFD), MNADK-null mice exhibited hepatic insulin resistance and glucose intolerance, indicating a type-2 diabetes-like phenotype in the absence of MNADK. MNADK deficiency led to a decrease in mitochondrial NADP(H) but an increase in cellular reactive oxygen species (ROS) in mouse livers. Consistently, protein levels of the major metabolic regulators or enzymes were decreased, while their acetylation modifications were increased in the livers of MNADK-null mice. Feeding mice with a HFD caused S-nitrosylation (SNO) modification, a posttranslational modification that represses protein activities, on MNADK protein in the liver. Reconstitution of an SNO-resistant MNADK variant, MNADK-S193, into MNADK-null mice mitigated hepatic steatosis induced by HFD. CONCLUSION: MNADK, the only known mammalian mitochondrial NAD kinase, plays important roles in preserving energy homeostasis to mitigate the risk of metabolic disorders.
Sujet(s)
Carcinome hépatocellulaire , Diabète de type 2 , Tumeurs du foie , Protéines mitochondriales , Stéatose hépatique non alcoolique , Phosphotransferases (Alcohol Group Acceptor) , Animaux , Diabète de type 2/complications , Diabète de type 2/génétique , Humains , Souris , Souris knockout , Mitochondries/enzymologie , Protéines mitochondriales/métabolisme , NAD/métabolisme , NADP/métabolisme , Stéatose hépatique non alcoolique/étiologie , Phosphotransferases (Alcohol Group Acceptor)/métabolismeRÉSUMÉ
NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.
Sujet(s)
Lysine , NAD , Acétylation , Domaine catalytique , Humains , Lysine/métabolisme , Protéines mitochondriales/métabolisme , NAD/métabolisme , NADP/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/métabolisme , Proline/métabolismeRÉSUMÉ
All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-Å structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization element, which is conserved in mitochondrial kinases of animals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.
Sujet(s)
Mitochondries , Protéines mitochondriales , NAD , Phosphotransferases (Alcohol Group Acceptor) , Multimérisation de protéines , Animaux , Humains , Mitochondries/enzymologie , Protéines mitochondriales/composition chimique , Protéines mitochondriales/métabolisme , NADP/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/composition chimique , Phosphotransferases (Alcohol Group Acceptor)/métabolismeRÉSUMÉ
Mitochondrial NAD kinase deficiency (NADK2D, OMIM #615787) is a rare autosomal recessive disorder of NADPH biosynthesis that can cause hyperlysinemia and dienoyl-CoA reductase deficiency (DECRD, OMIM #616034). NADK2 deficiency has been reported in only three unrelated patients. Two had severe, unremitting disease; one died at 4 months and the other at 5 years of age. The third was a 10 year old female with CNS anomalies, ataxia, and incoordination. In two cases mutations in NADK2 have been demonstrated. Here, we report the fourth known case, a 15 year old female with normal intelligence and a mild clinical and biochemical phenotype presumably without DECRD. Her clinical symptoms, which are now stable, became evident at the age of 9 with the onset of decreased visual acuity, bilateral optic atrophy, nystagmus, episodic lower extremity weakness, peripheral neuropathy, and gait abnormalities. Plasma amino acid levels were within normal limits except for mean lysine and proline levels that were 3.7 and 2.5 times the upper limits of normal. Whole exome sequencing (WES) revealed homozygosity for a g.36241900 A>G p. Met1Val start loss mutation in the primary NADK2 transcript (NM_001085411.1) encoding the 442 amino acid isoform. This presumed hypomorphic mutation has not been previously reported and is absent from the v1000GP, EVS, and ExAC databases. Our patient's normal intelligence and stable disease expands the clinical heterogeneity and the prognosis associated with NADK2 deficiency. Our findings also clarify the mechanism underlying NADK2 deficiency and suggest that this disease should be ruled out in cases of hyperlysinemia, especially those with visual loss, and neurological phenotypes.
Sujet(s)
Gènes de mitochondrie , Études d'associations génétiques , Protéines mitochondriales/déficit , Protéines mitochondriales/génétique , Mutation , Phénotype , Phosphotransferases (Alcohol Group Acceptor)/déficit , Phosphotransferases (Alcohol Group Acceptor)/génétique , Adolescent , Allèles , Séquence d'acides aminés , Substitution d'acide aminé , Marqueurs biologiques , Encéphale/anatomopathologie , Analyse de mutations d'ADN , Femelle , Génotype , Humains , Imagerie par résonance magnétique , Mâle , Protéines mitochondriales/composition chimique , Protéines mitochondriales/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/composition chimique , Phosphotransferases (Alcohol Group Acceptor)/métabolismeRÉSUMÉ
NAD kinase2 (NADK2) plays key roles in chloroplastic NADP biosynthesis, stress adaptation and modulation of cellular metabolisms in Arabidopsis. However, it is unknown whether and how NADK2 affects abscisic acid (ABA)-mediated stomatal movement. Here, we detected that null mutant nadk2 was more sensitive to drought stress than WT, and NADK2 gene was active in guard cells. Furthermore, NADK2 mutation impaired ABA-induced stomatal closure and ABA inhibition of light-promoted stomatal opening. NADK2 disruption also impaired ABA-stimulated accumulation of H2O2, Ca2+ and nitric oxide (NO) in guard cells, but did not affect the stomatal closure evoked by exogenous H2O2, Ca2+ or NO. Expression analysis revealed that ABA-promoted increases in transcripts of AtrbohD, AtrbohF and NIA1 were markedly arrested in guard cells of nadk2 compared with those of WT. Besides, genetic evidence indicated that NADK2 acted synergistically with OST1 and ABI1 during ABA-induced stomatal closure. Together, these results suggest that NADK2 is an essential positive regulator, and functions upstream of H2O2 in guard cell ABA signaling. It stimulates stomatal closure mainly through increasing the generation of H2O2, Ca2+ and NO in guard cells in Arabidopsis.