Critical Role In Cellular Metabolism Research Articles

Discovery and functional characterisation of protein CoAlation and the antioxidant function of coenzyme A

Coenzyme A (CoA) is an essential cofactor in all living cells which plays critical role in cellular metabolism, the regulation of gene expression and the biosynthesis of major cellular constituents. Recently, CoA was found to function as a major antioxidant in both prokaryotic and eukaryotic cells. This unconventional function of CoA is mediated by a novel post-translational modification, termed protein CoAlation. This review will highlight the history of this discovery, current knowledge, and future directions on studying molecular mechanisms of protein CoAlation and whether the antioxidant function of CoA is associated with pathologies, such as neurodegeneration and cancer.

Targeting the ATP-Axis in Lungs as a New Therapheutic Modality for COPD

Adenosine 5’-triphosphate (ATP) is found in every cell of the body where it plays a critical role in cellular metabolism and energetics. ATP is released from cells under physiologic and pathophysiologic conditions; extracellular ATP acts as an autocrine and paracrine agent. Its effects on targeted cells are mediated by subtypes of purinergic receptors (P2R). In the lungs, relatively large amounts of ATP are released under inflammatory conditions. Extracellular ATP triggers a central vagal reflex by activating purinergic receptor P2XR localized on pulmonary vagal sensory nerve terminals. This results in cough, bronchoconstriction and the release of pro-inflammatory neuropeptides via axon reflex. COPD patients manifest higher sensetivity to aerosolized ATP than healthy subjects, and the levels of ATP in COPD patients’ lungs are 3x that found in healthy subjects. This review succinctly details (i) the sources amd mechanisms of ATP’s release into the extracellular space, (ii) the ways extracellular ATP is eliminated, (iii) the deleterious effects of ATP in the lungs in general and in COPD in particular, and (iv) the rationale for the blockade of these actions of ATP in the lungs as a novel therapeutic approach in the management of COPD patients.

Enhanced production of iturin A by strengthening fatty acid synthesis modules in Bacillus amyloliquefaciens.

Iturin A is a biosurfactant with various applications, and its low synthesis capability limits its production and application development. Fatty acids play a critical role in cellular metabolism and target product syntheses, and the relationship between fatty acid supplies and iturin A synthesis is unclear. In this study, we attempted to increase iturin A production via strengthening fatty acid synthesis pathways in Bacillus amyloliquefaciens. First, acetyl-CoA carboxylase AccAD and ACP S-malonyltransferase fabD were overexpressed via promoter replacement, and iturin A yield was increased to 1.36 g/L by 2.78-fold in the resultant strain HZ-ADF1. Then, soluble acyl-ACP thioesterase derived from Escherichia coli showed the best performance for iturin A synthesis, as compared to those derived from B. amyloliquefaciens and Corynebacterium glutamicum, the introduction of which in HZ-ADF1 further led to a 57.35% increase of iturin A yield, reaching 2.14 g/L. Finally, long-chain fatty acid-CoA ligase LcfA was overexpressed in HZ-ADFT to attain the final strain HZ-ADFTL2, and iturin A yield reached 2.96 g/L, increasing by 6.59-fold, and the contents of fatty acids were enhanced significantly in HZ-ADFTL2, as compared to the original strain HZ-12. Taken together, our results implied that strengthening fatty acid supplies was an efficient approach for iturin A production, and this research provided a promising strain for industrial production of iturin A.

Abstract 2336: Pan-cancer analysis of NAMPT inhibitors reveals unique sensitivities to multiple NAMPT inhibitors in several cancer types

Abstract Nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in cellular metabolism and as such has been an active target for cancer drug therapy. NAMPT inhibition, via small molecule NAMPT inhibitors, disrupts cellular energy metabolism as well as other NAD+-dependent processes and has been shown to suppress cancer cell proliferation. The early clinical translation of NAMPT inhibitors has been hindered by toxicity, therefore the successful development of newer classes of NAMPT inhibitors will depend on identifying cancers with unique sensitivity to, and molecular biomarkers of susceptibility to NAMPT inhibition. Through our pan-cancer screens using several different NAMPT inhibitors on molecularly characterized cancer cell line panels, we have identified specific cancer types and subtypes that are consistently sensitive to NAMPT inhibition. In order to determine the efficacy of NAMPT inhibitors in different cancer cells, we performed proliferation assays on a large and fully characterized panel of human cancer cell lines (n=496) for response to several NAMPT inhibitors including FK866, KPT9274 and LSN3154567. Briefly, cells were treated with six dilutions of these inhibitors in duplicate, counted following a 6-day treatment window and IC50 values were determined for each cell line. Reverse-phase protein array (RPPA) analysis, comparative genomic hybridization (CGH), point mutation and RNAseq data for each cell line were then queried for correlation with NAMPT sensitivity/resistance. Proliferation assays indicate a strong correlation between sensitive and resistant cancer cell types to all three compounds despite their different mechanisms of action. Most notably, acute myeloid leukemia (AML), ovarian cancer, Ewing’s sarcoma and small cell lung cancer cells (SCLC) consistently demonstrated sensitivity to treatment with NAMPT inhibitors. Additional analysis revealed several baseline genomic and proteomic markers that were strongly associated with response to NAMPT inhibition. Our screening process provides a comprehensive overview of the cancer types and subtypes that demonstrate sensitivity to NAMPT inhibition. Furthermore, the molecular predictors of response identified in our screen may ultimately be useful in developing diagnostic tools for enrollment in biomarker-enriched clinical trials aimed at determining the potential use of NAMPT inhibitors. These results suggest the inhibitors’ therapeutic potential in patient populations presenting with AML, ovarian cancer, Ewing’s sarcoma and SCLC all of which currently have high unmet needs. Citation Format: Jenny J. Hong, Martina S. McDermott, Jewel K. Ng, Prita Pandya, Dennis J. Slamon. Pan-cancer analysis of NAMPT inhibitors reveals unique sensitivities to multiple NAMPT inhibitors in several cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2336.

STALLION: a stacking-based ensemble learning framework for prokaryotic lysine acetylation site prediction.

Protein post-translational modification (PTM) is an important regulatory mechanism that plays a key role in both normal and disease states. Acetylation on lysine residues is one of the most potent PTMs owing to its critical role in cellular metabolism and regulatory processes. Identifying protein lysine acetylation (Kace) sites is a challenging task in bioinformatics. To date, several machine learning-based methods for the in silico identification of Kace sites have been developed. Of those, a few are prokaryotic species-specific. Despite their attractive advantages and performances, these methods have certain limitations. Therefore, this study proposes a novel predictor STALLION (STacking-based Predictor for ProkAryotic Lysine AcetyLatION), containing six prokaryotic species-specific models to identify Kace sites accurately. To extract crucial patterns around Kace sites, we employed 11 different encodings representing three different characteristics. Subsequently, a systematic and rigorous feature selection approach was employed to identify the optimal feature set independently for five tree-based ensemble algorithms and built their respective baseline model for each species. Finally, the predicted values from baseline models were utilized and trained with an appropriate classifier using the stacking strategy to develop STALLION. Comparative benchmarking experiments showed that STALLION significantly outperformed existing predictor on independent tests. To expedite direct accessibility to the STALLION models, a user-friendly online predictor was implemented, which is available at: http://thegleelab.org/STALLION.

Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Creatine in Health and Disease.

Although creatine has been mostly studied as an ergogenic aid for exercise, training, and sport, several health and potential therapeutic benefits have been reported. This is because creatine plays a critical role in cellular metabolism, particularly during metabolically stressed states, and limitations in the ability to transport and/or store creatine can impair metabolism. Moreover, increasing availability of creatine in tissue may enhance cellular metabolism and thereby lessen the severity of injury and/or disease conditions, particularly when oxygen availability is compromised. This systematic review assesses the peer-reviewed scientific and medical evidence related to creatine’s role in promoting general health as we age and how creatine supplementation has been used as a nutritional strategy to help individuals recover from injury and/or manage chronic disease. Additionally, it provides reasonable conclusions about the role of creatine on health and disease based on current scientific evidence. Based on this analysis, it can be concluded that creatine supplementation has several health and therapeutic benefits throughout the lifespan.

Nicotinamide and NAFLD: Is There Nothing New Under the Sun?

Nicotinamide adenine dinucleotide (NAD) has a critical role in cellular metabolism and energy homeostasis. Its importance has been established early with the discovery of NAD’s therapeutic role for pellagra. This review addresses some of the recent findings on NAD physiopathology and their effects on nonalcoholic fatty liver disease (NAFLD) pathogenesis, which need to be considered in the search for a better therapeutic approach. Reduced NAD concentrations contribute to the dysmetabolic imbalance and consequently to the pathogenesis of NAFLD. In this perspective, the dietary supplementation or the pharmacological modulation of NAD levels appear to be an attractive strategy. These reviewed studies open the doors to growing interest in NAD metabolism for NAFLD diagnosis, prevention, and treatment. Future rigorous clinical studies in humans will be necessary to validate these preliminary but promising results.

Clinical characterization of patients with SDHC epimutation in gastrointestinal stromal tumors.

11033 Background: Gastrointestinal Stromal Tumors are the most common malignancy in the GI tract. While the vast majority exhibit somatic mutations in KIT and PDGFRA, approximately 15% of GIST patients do not have this feature. This group of KIT and PDGFRA “wildtype” GISTs have in common a negative expression of SDHB when interrogated by immunohistochemistry. Succinate dehydrogenase (SDH) is a conserved enzyme that plays a critical role in cellular metabolism and energy production. A loss in SDH function is a mechanism observed in several types of cancers, and germline SDH mutations are considered a tumor predisposition syndrome. This group has reported that SDH-deficient gastrointestinal stromal tumors often harbor germline mutations in the SDH subunit genes (SDHA, SDHB, SDHC, and SDHD, termed SDHx). There is, however, a defined group that shows lack of SDH expression in the absence of mutation. Methods: We performed targeted exome sequencing on GIST patients’ tumor samples from the NIH GIST clinic and identified 25 SDHx-WT cases. Genome-wide DNA methylation and expression profiling showed SDHC promoter-specific CpG island hypermethylation and gene silencing in these 25 SDHx-WT, SDH deficient GISTs. Results: Clinical characterization of this cohort revealed that 24 of 25 SDHC-epimutant GISTs occurred in female patients, with a median age of 12 upon diagnosis. The median tumor size of this cohort of patients was 4.0 cm. Of the 16 patients from whom we were able to obtain complete pathology data, 15 showed epithelioid or mixed-epithelioid tumor morphology. All of them showed negative immunohistochemical staining for SDHB. 15 of 16 patients had multifocal tumors, which is a common finding in this population. Conclusions: The profiling of this cohort provides further insights into the natural history and pathogenesis of SDHC-epimutant GIST tumors.

NAMPT expression in stage I/II pancreatic ductal adenocarcinoma.

e15735 Background: NAD (nicotinamide adenine dinucleotide), a vital cofactor in redox reactions and a substrate for many enzymes, plays a critical role in cellular metabolism. NAMPT (nicotinamide phosphoribosyltransferase) is a key enzyme that regenerates NAD from its metabolites. NAMPT has been shown to be overexpressed in some cancers, and inhibition of NAMPT in pancreas cancer cell lines and xenografts has been shown to decrease cell growth, making it an attractive potential therapeutic target. Here we correlate NAMPT expression in a large cohort of stage I/II pancreatic ductal adenocarcinomas (PDA) with clinical outcomes and pathologic features. Methods: NAMPT immunohistochemistry (IHC) was performed on tissue microarrays of 250 PDAs. Tumors had been arrayed in triplicate. Each core was evaluated for staining intensity of cancer cells (0 = no staining, 1+ = weak, 2+ = moderate, 3+ = strong), and a mean score was calculated for each case; cases with fewer than 2 cores were excluded. Fisher’s exact and Wilcoxon rank sum tests were used for categorical and continuous variables, respectively. Recurrence-free survival (RFS) and overall survival (OS) differences were evaluated using Cox regression. This study was conducted under an approved IRB protocol. Results: 197 PDAs had at least 2 TMA cores with identifiable cancer cells. The mean IHC score was 0.53 (range 0 to 2.33). While 121/197 PDAs (61%) were positive, only 18/197 (9%) had a score > 1. Stage II tumors had a higher mean score than stage I tumors (0.57 vs 0.31; P = 0.03) and were more likely to be positive (66% of 148 vs 41% of 22; P = 0.03). However, RFS and OS were not significantly different between NAMPT-positive vs NAMPT-negative PDAs. Conclusions: NAMPT expression was detected in 61% of stage I/II PDAs. Frequency and extent of expression correlated with overall stage, suggesting a role in cancer progression. Although NAMPT expression did not correlate with RFS and OS in this study, given its frequent expression in PDA, it represents a potential therapeutic target.

Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role for 14-3-3ε in Metabolic Pathways.

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.

Screening SIRT1 Activators from Medicinal Plants as Bioactive Compounds against Oxidative Damage in Mitochondrial Function.

Sirtuin type 1 (SIRT1) belongs to the family of NAD+ dependent histone deacetylases and plays a critical role in cellular metabolism and response to oxidative stress. Traditional Chinese medicines (TCMs), as an important part of natural products, have been reported to exert protective effect against oxidative stress in mitochondria. In this study, we screened SIRT1 activators from TCMs and investigated their activities against mitochondrial damage. 19 activators were found in total by in vitro SIRT1 activity assay. Among those active compounds, four compounds, ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were further studied to validate the SIRT1-activation effects by liquid chromatography-mass spectrometry and confirm their activities against oxidative damage in H9c2 cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). The results showed that those compounds enhanced the deacetylated activity of SIRT1, increased ATP content, and inhibited intracellular ROS formation as well as regulating the activity of Mn-SOD. These SIRT1 activators also showed moderate protective effects on mitochondrial function in t-BHP cells by recovering oxygen consumption and increasing mitochondrial DNA content. Our results suggested that those compounds from TCMs attenuated oxidative stress-induced mitochondrial damage in cardiomyocytes through activation of SIRT1.

Combination of Cetuximab and Rapamycin Enhances the Therapeutic Efficacy in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide. It is well known that the activation of PI3K/AKT/mTOR and the Ras/MAPK signaling pathway plays a critical role in cellular metabolism, growth and proliferation, and its inhibitors have been used as therapeutic drugs for hepatocellular carcinoma. Cetuximab, a chimerical monoclonal EGFR IgG1 antibody, can block the binding of EGF or other ligands to EGFR and thus inhibit ligands-induced receptor phosphorylation. In the present study, we found that rapamycin could enhance the antiproliferation effect of cetuximab in both HepG2 cells and Huh-7 cells and arrest the cell cycle. Cetuximab in combination with rapamycin had synergistic effects on inhibiting the phosphrylation of proteins in PI3K/AKT/mTOR and Ras/MAPK signaling pathway. Combination of cetuximab with rapamycin treatment significantly suppressed the HCC development in HepG2 cells-xenografted mice and improved the survival. Cetuximab and rapamycin inhibited the growth of HCC both in vitro and in vivo. These results suggest that the combination therapy using the inhibitors for both EGFR and PI3K/AKT/mTOR signaling pathways may be a novel therapeutic approach for HCC.

Insulin for the treatment of hyperkalemia: a double-edged sword?

Potassium plays a critical role in cellular metabolism and normal neuromuscular function. Tightly regulated homeostatic mechanisms have developed in the process of evolution to provide primary defense against the threats of hyper- and hypokalemia. The kidney plays a primary role in potassium balance, by increasing or decreasing the rate of potassium excretion. Distribution of potassium between the intracellular and the extracellular fluid compartments is regulated by physiologic factors such as insulin and catecholamines which stimulate the activity of the Na+-K+ ATPase. Only about 10% of the ingested potassium is excreted via the gut under normal physiologic conditions [1].

The mTOR pathway in hepatic malignancies

The mechanistic/mammalian target of rapamycin (mTOR) pathway plays a critical role in cellular metabolism, growth, and proliferation and has been evaluated as a target for therapy in various malignancies. The mTOR pathway is a major tumor-initiating pathway in hepatocellular carcinoma, with up-regulation seen in up to 50% of tumors. Metformin, which represses mTOR signaling by activating adenosine monophosphate-activated protein kinase, has been shown to decrease liver carcinogenesis in population studies. mTOR inhibitors such as everolimus have been evaluated as adjunctive chemotherapy with some success, although efficacy has been limited by the lack of complete mTOR pathway inhibition. The active site mTOR inhibitors hold greater promise, given that they offer complete mTOR suppression. There is also evidence of mTOR pathway activation in cholangiocarcinoma, although its biological significance in initiating and promoting tumor progression remains ambiguous. This review provides an overview of the complex biochemistry behind the mTOR pathway and its role in carcinogenesis, especially as it pertains to hepatic malignancies.

Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

Role of SIRT1 in regulation of hepatic gene expression by thyroid hormone

Thyroid hormone induces expression of hepatic genes such as pyruvate dehydrogenase kinase 4 (PDK4) and carnitine palmitoyltransferase1a (CPT1a) through the thyroid hormone receptor. Peroxisome proliferator‐activated receptor gamma coactivator 1 (PGC‐1α) enhances the thyroid hormone (T3) induction of these genes. The NAD‐dependent sirtuin 1 (SIRT1) deacetylase plays a critical role in cellular metabolism by deacetylating and activating transcription factors including PGC‐1α. The SIRT1 activator, resveratrol, improves mitochondrial function, increases fatty acid oxidation and decreases lipogenesis. Here, we investigated the role of SIRT1 in the T3 regulation of PDK4 and CPT1a genes. Our data indicate that resveratrol increases expression of PDK4 and CPT1a genes in the liver and enhances the T3 induction of these genes. Resveratrol enhanced the T3 induction through the gene promoters. Overexpression of SIRT1 has similar effects to resveratrol whereas knockdown of SIRT 1 reduces the T3 induction of PDK4 and CPT1a. Moreover, SIRT1 interacts with the thyroid hormone receptor β. Our data indicate that SIRT1 plays an important role in the thyroid hormone induction of hepatic genes such as PDK4 and CPT1a.This work was supported by National Institute of Health and American Heart Association.

Mitochondrial Dynamics in Heart Failure

Mitochondria have been widely studied for their critical role in cellular metabolism, energy production, and cell death. New developments in research on mitochondria derived from studies in yeast have led to the discovery of entirely new mitochondrial processes that have implications for mitochondrial function in heart failure. Recent studies have identified that maintaining normal mitochondrial morphology and function depends on the dynamic balance of mitochondrial fusion and fission (division). Mitochondrial fusion and fission are constant ongoing processes, which are essential for the maintenance of normal mitochondrial function. Studies in heart failure have been limited but suggest a possible reduction in mitochondrial fusion. As mitochondrial fusion and fission have important links to apoptosis, a key mechanism of loss of cardiac myocytes in heart failure, there are many implications for both heart failure research and treatment.

Mitochondrial dysfunction and oxidative status impairment: A comparative study between male and female 3xTg-AD mice

Alzheimerś disease (AD) is a progressive and fatal neurodegenerative disorder where, besides aging, gender is an established risk factor. Indeed, epidemiological studies show that women have a higher prevalence and risk for AD. Mitochondria are intracellular organelles that despite playing a critical role in cellular metabolism are also one of the major physiological sources of reactive oxygen species. Evidence also shows that mitochondrial dysfunction and oxidative stress are underlying factors in several neurodegenerative disorders including AD. The 3xTg-AD mouse is an animal model for AD that develops an age-related and progressive neuropathological phenotype that includes both plaque and tangle pathology.The 3xTg-AD mouse is an animal model for AD that develops an age-related and progressive neuropathological phenotype that includes both plaque and tangle pathology. In this study we sought to evaluate gender effects in cortical and hippocampal mitochondria bioenergetics and oxidative status of 9-month-old male and female 3xTg-AD mice. We evaluated respiratory chain and phosphorylation system parameters, hydrogen peroxide (H2O2) production rate, mitochondrial aconitase activity and enzymatic and non-enzymatic antioxidant defenses. Results show that cortical and hippocampal mitochondria from female mice possess a significant increase in aconitase activity compared to male mice. Cortical mitochondria from female mice also present a slight increase in ADP/O index and a decrease in H2O2 production when compared to male mice. Additionally, hippocampal mitochondria from 3xTg-AD males show a significant decrease in states 3 and 4 of respiration and in aconitase activity when compared to male control mice. In turn, cortical mitochondria from 3xTg-AD males present a significant increase in H2O2 production rate compared to control males. In hippocampal mitochondria from female 3xTg-AD mice a significant decrease in aconitase activity was observed. Our results show that, at this age, 3xTg-AD males are more prone to mitochondrial dysfunction and oxidative stress, which may exacerbate the neurodegenerative events.

Identification of NAD interacting residues in proteins

BackgroundSmall molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD) is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP) have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs) from amino acid sequence using bioinformatics tools.ResultsWe extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His) in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM) based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC) 0.47 with accuracy 74.13% at window length 17. We also developed a SVM based method using evolutionary information in the form of position specific scoring matrix (PSSM) and obtained maximum MCC 0.75 with accuracy 87.25%.ConclusionFor the first time a sequence-based method has been developed for the prediction of NAD binding proteins and their interacting residues, in the absence of any prior structural information. The present model will aid in the understanding of NAD+ dependent mechanisms of action in the cell. To provide service to the scientific community, we have developed a user-friendly web server, which is available from URL http://www.imtech.res.in/raghava/nadbinder/.