Nicotinamide Adenine Dinucleotide Binding Research Articles

The T120P or M172V mutation on rv2172c confers high level para-aminosalicylic acid resistance in Mycobacterium tuberculosis

Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172 V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172 V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172 V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172 V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.

Multi-omics reveals new links between Fructosamine-3-Kinase (FN3K) and core metabolic pathways

Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the Tree of Life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) HepG2 cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), and carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3K and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. Our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance and NAD-dependent metabolic processes are altered.

An in silico approach to investigate the theranostic potential of coumarin-derived self-immolative luminescent probes.

Till date the challenge exists in the treatments of cancer for various reasons. Most importantly, the available diagnostics are expensive with research gap for enhancing the cancer detection sensitivity. Herein, a series of coumarin-derived fluorescent theranostic probes are reported that can serve as potent anticancer agents as well as in the detection of cancer cells. The potential of these probes to efficiently block one of the well-known cancer drug targets NADPH quinone oxidoreductase-1 (NQO1) is evaluated through various pharmacokinetic methods including absorption, distribution, metabolism and excretion (ADME) properties evaluation, PASS (prediction of activity spectra for substance) algorithm along with molecular docking and dynamic simulations. Further the luminescent properties of these molecules were evaluated by investigating their electronic properties in the ground and excited states with the help of density functional theory methods. Results indicate that the proposed molecules can potentially block the NADPH (reduced form of nicotinamide adenine dinucleotide) binding site of NQO1, thereby inhibiting the activity of the enzyme to ultimately disrupt the metabolism of cancer cells.

Proteomic signatures of infiltrative gastric cancer by proteomic and bioinformatic analysis.

Proteomic signatures of Ming's infiltrative gastric cancer (IGC) remain unknown. To elucidate the molecular characteristics of IGC at the proteomics level. Twelve pairs of IGC and adjacent normal tissues were collected and their proteomes were analyzed by high performance liquid chromatography tandem mass spectrometry. The identified peptides were sequenced de novo and matched against the SwissProt database using Maxquant software. The differentially expressed proteins (DEPs) were screened using |log2(Fold change)| > 1 and P-adj < 0.01 as the thresholds. The expression levels of selected proteins were verified by Western blotting. The interaction network of the DEPs was constructed with the STRING database and visualized using Cytoscape with cytoHubba software. The DEPs were functionally annotated using clusterProfiler, STRING and DAVID for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. P < 0.05 was considered statistically significant. A total of 7361 DEPs were identified, of which 94 were significantly up-regulated and 223 were significantly down-regulated in IGC relative to normal gastric tissues. The top 10 up-regulated proteins were MRTO4, BOP1, PES1, WDR12, BRIX1, NOP2, POLR1C, NOC2L, MYBBP1A and TSR1, and the top 10 down-regulated proteins were NDUFS8, NDUFS6, NDUFA8, NDUFA5, NDUFC2, NDUFB8, NDUFB5, NDUFB9, UQCRC2 and UQCRC1. The up-regulated proteins were enriched for 9 biological processes including DNA replication, ribosome biogenesis and initiation of DNA replication, and the cellular component MCM complex. Among the down-regulated proteins, 17 biological processes were enriched, including glucose metabolism, pyruvic acid metabolism and fatty acid β-oxidation. In addition, the mitochondrial inner membrane, mitochondrial matrix and mitochondrial proton transport ATP synthase complex were among the 6 enriched cellular components, and 11 molecular functions including reduced nicotinamide adenine dinucleotide dehydrogenase activity, acyl-CoA dehydrogenase activity and nicotinamide adenine dinucleotide binding were also enriched. The significant KEGG pathways for the up-regulated proteins were DNA replication, cell cycle and mismatch repair, whereas 18 pathways including oxidative phosphorylation, fatty acid degradation and phenylalanine metabolism were significantly enriched among the down-regulated proteins. The proteins involved in cell cycle regulation, DNA replication and mismatch repair, and metabolism were significantly altered in IGC, and the proteomic profile may enable the discovery of novel biomarkers.

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Integrated In Silico Studies on the Role of Nicotinamide Adenine Dinucleotide (NADH) Binding in Activating C-Terminal Binding Protein 2 (CtBP2)

Abstract The C-terminal binding protein 2 (CtBP2) plays a role in apoptosis and embryogenesis. Genetic knockout studies have been demonstrated to cause severe developmental defects and embryonic lethality. CtBP2 has three key domains and dimerizes for a significant role by binding with nicotinamide adenine dinucleotide (NADH). However, the molecular mechanism of CtBP2 dimerization and the effect of NADH binding are unknown. In this study, we performed molecular dynamics (MD) and docking simulation to reveal the mechanism of C-terminal binding protein 2 (CtBP2) dimerization and the effect of NADH binding on the dimer formation. Our MD simulation results detected seven salt bridges that are important for CtBP2 dimerization. And docking simulation demonstrated that the holo-monomer gave a higher probability of correct docking pose than the apo-monomer. Moreover, in docking simulation using a PXDLS model peptide, that holo form gave more docking poses than that apo form. These results suggested that the holo form has a structure that facilitates the formation of dimers and the binding of PXDLS peptides.

A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton’s Tyrosine Kinase Inhibitor CC-292

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.

NADPH oxidase involved in immune response via regulating the expression of antioxidant genes in Litopenaeus vannamei

Free radicals such as reactive oxygen species (ROS) play a significant role in the innate immune system. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) are found to be involved in ROS production and played crucial roles in host biological processes of humans, insects, and plants. However, there are relatively few studies on the function of Noxes in crustaceans. In this study, the full-length cDNA of Nox was cloned from Litopenaeus vannamei by rapid amplification of cDNA ends (RACE) to obtain a 3790 base pairs (bp) cDNA sequence. LvNox cDNA comprised a 5’-untranslated region (UTR) of 454 bp, a 3’-UTR of 333 bp, and a putative open reading frame (ORF) of 3006 bp. Structural analysis showed that LvNox had a transmembrane helical segment (THS) at positions 233-255, a flavin adenine dinucleotide (FAD) binding domain (at positions 447-579), and a nicotinamide adenine dinucleotide (NAD) binding domain (at positions 815-990). Tissue distribution analysis revealed that LvNox mRNA was expressed in heart, intestine, muscle, gill, hepatopancreas, stomach, epidermis, hemocyte, and eyestalk. High expression levels of LvNox were observed in epidermis, hemocyte and eyestalk. The expression levels of LvNox were significantly induced after Vibrio alginolyticus injection for 6, 12, 24 and 48 h, and after Cu exposure for 24 and 48 h. Furthermore, the RNA interference of LvNox significantly reduced the production of ROS and the expression levels of copper/zinc superoxide dismutase (Cu-Zn SOD), catalase (CAT) and nitric oxide synthase (NOS). These results indicated that LvNox could be induced by V. alginolyticus infection and Cu exposure, which might be involved in immune response of shrimp via regulating the expression of antioxidant genes. These findings provide a solid foundation for further study of the function of Noxes in crustaceans.

Dual Oxidase System Genes Defects in Children With Congenital Hypothyroidism.

The objectives of this study were to analyze the distribution of dual oxidase (DUOX) system genes (containing DUOX2, DUOX1, DUOXA2, and DUOXA1) variants in children with congenital hypothyroidism (CH) and their phenotypes. Target region sequencing technology was performed on DUOX system genes among 606 CH subjects covering all the exon and intron regions. Detailed clinical data were collected for statistical analysis. A total of 95 suspected pathogenic variants were detected in the DUOX system genes, showing a 39.11% rate in variant carrying (237/606). DUOX2 had the highest rate in this study. There were statistical differences in maximum adjusted dose and current dose of levothyroxine between the DUOX system genes nonmutated group with the mutated group (both Ps < 0.001). The cases in the DUOX system genes mutated group were more likely to develop into transient CH (χ 2 = 23.155, P < 0.001) and more likely to manifested as goiter or gland-in-situ (χ 2 = 66.139, P < 0.001). In addition, there was no significant difference in clinical characteristics between DUOX system genes monoallelic and non-monoallelic. Although 20% of the variants affected the functional domain regions (EF hand, flavin adenine dinucleotide and nicotinamide adenine dinucleotide binding sites), there was no significant effect on the phenotype severity whether the variation is located in the functional domain regions. Our results showed the high variation rate of DUOX2 in the DUOX system genes among Chinese CH patients. The complex genotype-phenotype relationship of DUOX system genes broadened the understanding of CH phenotype spectrum.

Highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives and in silico interaction analysis with putative target Plasmodium falciparum lactate dehydrogenase

Malaria infection caused by Plasmodium falciparum is majorly responsible for millions of deaths in humans every year. Moreover, a rapid increase in resistance to existing drugs has posed an urgent need for new anti-malarials. Herein, we report the highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives, inspired from naturally occurring dependensin against chloroquine (CQ) sensitive and resistant P. falciparum strains. Chemically synthesized, four dependensin analogs 85(A–D) exhibited growth inhibition at nanomolar concentrations ranging from 63.96 to 725.8 nM by blocking the parasite development at the ring and early trophozoite stages. The growth inhibitory activity of dependensin analogs was correlated with their anti-plasmodial lactate dehydrogenase activity by computational analysis. Molecular docking, 50 ns simulation and a 2D-Quantitative Structure-Activity Relationship (2D-QSAR) modelling revealed the interaction with their putative target P. falciparum lactate dehydrogenase (PfLDH). Here, developing the predictive 2D descriptors such as thermodynamic, spatial, electronic, and topological with multiple linear regression analysis (MLRA), the structural requirements for potent and selective PfLDH inhibitory activity has been identified. The strong binding of compound 85D to the catalytic Nicotinamide adenine dinucleotide (NADH) binding pocket of the PfLDH further supported the PfLDH targeting potential of dependensin analogs. Overall, this study revealed a highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives with their putative anti-PfLDH activity. Communicated by Ramaswamy H. Sarma

Differentially expressed lncRNAs, miRNAs and mRNAs with associated ceRNA networks in a mouse model of myocardial ischemia/reperfusion injury.

Non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs), have significant regulatory effects on a number of biological processes in myocardial ischemia/reperfusion (I/R) injury, including cell differentiation, proliferation and apoptosis. In the present study, the expression levels of lncRNAs, miRNAs and mRNAs were evaluated in a mouse model of myocardial I/R injury. The potential functions of these differentially expressed genes were then analyzed via Gene Ontology and pathway analyses. Additionally, the interactions between lncRNA-miRNA-mRNA were predicted by constructing a competing endogenous RNA regulatory network. It was found that 14,366 lncRNAs, 151 miRNAs and 9,377 mRNAs were differentially expressed in mice hearts after I/R compared with the Sham group (fold change >2; P<0.05). The results indicated that these differentially expressed genes were involved in multiple molecular functions, including ‘guanosine diphosphate binding’, ‘RNA polymerase II carboxy-terminal domain kinase activity’, ‘TATA-binding protein-class protein binding’, ‘nicotinamide adenine dinucleotide binding’ and ‘protein phosphatase type 2A regulator activity’. The interactions between lncRNA-miRNA-mRNA, including five lncRNAs, 38 miRNAs and 196 mRNAs, were predicted, specifically Gm12040-mmu-miR-125a-5p-decapping mRNA 1B, Rpl7l1-ps1-mmu-miR-124-3p-G protein-coupled receptor 146, Gm11407-mmu-miR-190a-5p-homeobox and leucine zipper encoding (HOMEZ), 1600029O15Rik-mmu-miR-132-3p-HOMEZ and AK155692-mmu-miR-1224-3p-activating transcription factor 6β. Collectively, these findings provided novel insights for future research on lncRNAs, miRNAs and mRNAs in myocardial I/R injury.

MoNFR, encoding a putative NADPH-ferrihemoprotein reductase, is required for the pathogenicity of Magnaporthe oryzae

A wild type Magnaporthe oryzae CY2 with normal growth and sporulation but weak pathogenicity is not virulent to more than over 1000 rice cultivars. Based on the transformant library of M. oryzae CY2 by Agrobacterium tumefaciens-mediated transformation (ATMT), we screened a transformant T23 with normal infectious hyphae and pathogenicity to rice line IRBL-19. Co-segregation and southern blotting suggested that T-DNA is inserted with single copy, and pathogenicity may require the mutated gene MoNFR. Bioinformatics analysis predicted that the gene MoNFR encoded a putative NADPH-ferrihemoprotein reductase with an indole amine 2,3-dioxgyenase (IDO) domain, a flavin adenine dinucleotide binding domain (FAD_binding domain), a nicotinamide adenine dinucleotide binding domain (NAD_binding domain) and a cytochrome-b5 domain. Vectors were constructed to knock-out the target gene. Two mutants with MoNFR disruptant were obtained from the wild type strain CY2 by targeted gene replacement with hygromycin B phosphotransferase (HPH) gene. Compared with strain T23 and CY2, the MoNFR mutants showed no significant difference in colony morphology, growth rate and mycelium color but conidia produced in culture were 4.5–5 times more and higher percentage of appressorial and infectious hyphae formation was observed on onion epidermis. They retain the pathogenicity to IRBL-19, and are similar in virulence to strain T23. These results indicate that MoNFR plays a key role in the pathogenicity of M. oryzae.

Machilin A Inhibits Tumor Growth and Macrophage M2 Polarization Through the Reduction of Lactic Acid.

Lactate dehydrogenase A (LDHA) is an important enzyme responsible for cancer growth and energy metabolism in various cancers via the aerobic glycolytic pathway. Here, we report that machilin A (MA), which acts as a competitive inhibitor by blocking the nicotinamide adenine dinucleotide (NAD) binding site of LDHA, suppresses growth of cancer cells and lactate production in various cancer cell types, including colon, breast, lung, and liver cancers. Furthermore, MA markedly decreased LDHA activity, lactate production, and intracellular adenosine triphosphate (ATP) levels induced by hypoxia-induced LDHA expression in cancer cells, and significantly inhibited colony formation, leading to reduced cancer cell survival. In mouse models inoculated with murine Lewis lung carcinoma, MA significantly suppressed tumor growth as observed by a reduction of tumor volume and weight; resulting from the inhibition of LDHA activity. Subsequently, the suppression of tumor-derived lactic acid in MA-treated cancer cells resulted in decrease of neovascularization through the regulation of alternatively activated macrophages (M2) polarization in macrophages. Taken together, we suggest that the reduction of lactate by MA in cancer cells directly results in a suppression of cancer cell growth. Furthermore, macrophage polarization and activation of endothelial cells for angiogenesis were indirectly regulated preventing lactate production in MA-treated cancer cells.

NAD binding by human CD38 analyzed by Trp189 fluorescence

The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2′-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 μM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37 μM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Differential Responses of the Nitric Oxide Synthase (NOS) Gene to Immunological Challenges in Noble Scallop Chlamys nobilis

AbstractNitric oxide synthase (NOS) plays important roles in the immune system of animals. As an important marine bivalve cultured in south China, the noble scallop Chlamys nobilis is subject to diseases of various kinds, leading to decrease harvest. To investigate whether NOS in the noble scallop (hereafter, CnNOS) plays a significant role in immune defense, the full‐length open‐reading frame (ORF) of the CnNOS gene was cloned, and its expression levels in different tissues were determined. The complementary DNA nucleotide sequence of CnNOS encompassed 5,345 bp, with a full‐length ORF of 4,647 bp. The gene consisted of the typical NOS structure, including an NOS domain, a flavodoxin 1 domain, a flavin adenine dinucleotide binding domain, and a conservative nicotinamide adenine dinucleotide binding domain. Subsequently, transcript levels of CnNOS in hemocytes were determined by real‐time quantitative reverse transcription PCR, indicating that CnNOS expression was significantly up‐regulated in noble scallops injected with different immunostimulants, including lipopolysaccharides; the synthetic double‐stranded RNA, polyinosinic: polycytidylic acid; and Vibrio parahaemolyticus. The present results suggest that the CnNOS might play a crucial role in the noble scallop's defense against exogenous stimuli.

Proteomic analysis of melatonin-mediated osmotic tolerance by improving energy metabolism and autophagy in wheat (Triticum aestivum L.).

Melatonin-mediated osmotic tolerance was attributed to increased antioxidant capacity, energy metabolism, osmoregulation and autophagy in wheat (Triticum aestivum L.). Melatonin is known to play multiple roles in plant abiotic stress tolerance. However, its role in wheat has been rarely investigated. In this study, 25% polyethylene glycol 6000 (PEG 6000) was used to simulate osmotic stress, and wheat seeds and seedlings were treated with different concentrations of melatonin under PEG stress. Isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic techniques were used to identify the differentially accumulated proteins from melatonin-treated and non-treated seedlings. Seeding priming with melatonin significantly increased the germination rate, coleoptile length, and primary root number of wheat under PEG stress, as well as the fresh weight, dry weight, and water content of wheat seedlings. Under PEG stress, melatonin significantly improved reactive oxygen species homeostasis, as revealed by lower H2O2 and O 2· content; and the expression of antioxidant enzymes at the transcription and translation levels was increased. Melatonin maintained seedling growth by improving photosynthetic rates and instantaneous and intrinsic water use efficiencies, as well as carbon fixation and starch synthesis at the protein level. Melatonin treatment significantly affected the expression of glycolytic proteins, including fructose-1,6-bisphosphate aldolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and remarkably increased the expression of the nicotinamide adenine dinucleotide transporter and nicotinamide adenine dinucleotide binding protein, thereby indirectly modulating electron transport in the respiratory chain. This indicated that melatonin improved energy production in PEG-stressed seedlings. Further, melatonin played a regulatory role in autophagy, protease expression, and ubiquitin-mediated protein degradation by significantly upregulating rab-related protein, fused signal recognition particle receptor, aspartyl protease, serine protease, ubiquitin-fold modifier 1, and ubiquitin at the mRNA or protein level. These findings suggested that melatonin might activate a metabolic cascade related to autophagy under PEG stress in wheat seedlings.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+ Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach.

Protein-ligand interaction plays a critical role in regulating the biochemical functions of proteins. Discovering protein targets for ligands is vital to new drug development. Here, we present a strategy that combines experimental and computational approaches to identify ligand-binding proteins in a proteomic scale. For the experimental part, we coupled pulse proteolysis with filter-assisted sample preparation (FASP) and quantitative mass spectrometry. Under denaturing conditions, ligand binding affected protein stability, which resulted in altered protein abundance after pulse proteolysis. For the computational part, we used the software Patch-Surfer2.0. We applied the integrated approach to identify nicotinamide adenine dinucleotide (NAD)-binding proteins in the Escherichia coli proteome, which has over 4200 proteins. Pulse proteolysis and Patch-Surfer2.0 identified 78 and 36 potential NAD-binding proteins, respectively, including 12 proteins that were consistently detected by the two approaches. Interestingly, the 12 proteins included 8 that are not previously known as NAD binders. Further validation of these eight proteins showed that their binding affinities to NAD computed by AutoDock Vina are higher than their cognate ligands and also that their protein ratios in the pulse proteolysis are consistent with known NAD-binding proteins. These results strongly suggest that these eight proteins are indeed newly identified NAD binders.

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

Chromosomal damage and micronucleus induction by MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor: Evidence for a non-DNA-reactive mode of action

MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that competes with the binding of the PARP substrate nicotinamide adenine dinucleotide (NAD), is being developed as a neuroprotective agent against acute ischemic stroke. MP-124 increased structural chromosomal aberration in CHL/IU cells, but showed negative results in the bacterial reverse mutation test, and the rat bone marrow micronucleus (MN) and the rat liver unscheduled DNA synthesis tests after the intravenous bolus injection. Thus, MP-124 did not appear to be direct-acting mutagen. Since, PARP-1 is a key enzyme in DNA repair, the effect of continuous PARP-1 inhibition by MP-124 was further examined in the rat MN test under 24-h intravenous infusion, and an increase in micronucleated immature erythrocytes (MNIE) was observed. The increase was clearly reduced by co-treatment with nicotinic acid, which resulted in increased intracellular NAD levels. This is consistent with the established activity of MP-124 as a competitive inhibitor of PARP and provides strong evidence that the DNA-damaging effect that leads to the increase in MNIE is a secondary effect of PARP-1 inhibition. This mechanism is expected to result in a threshold for the induction of MNIE by MP-124, and allows for the establishment of a safe margin of exposure for the therapeutic use of MP-124.