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Neuropharmacology Feb 2023Purine-based molecules play ancient, fundamental, and evolutionarily-conserved roles across life on Earth, ranging from DNA and RNA, to the universal energy currency,... (Review)
Review
Purine-based molecules play ancient, fundamental, and evolutionarily-conserved roles across life on Earth, ranging from DNA and RNA, to the universal energy currency, ATP. In mammals, the two primary routes for the synthesis of the adenine nucleotides ATP, ADP and AMP, and, as a consequence, the major bioactive metabolite adenosine, are the de novo purine biosynthesis (DNPB) pathway, and the purine salvage pathway (PSP). Of the two, the PSP dominates in both the mammalian brain and heart. This is because the PSP utilizes the breakdown products of ATP, occasioned by the high energy demands of these organs, to rapidly regenerate adenine nucleotides. This resynthesis route, while efficient and energetically favourable, leaves these organs vulnerable to loss of salvageable metabolites, with the potential for protracted depletion of the means to synthesize ATP, and the ability to deploy neuro- and cardioprotective adenosine. Having previously shown that hippocampal cellular ATP and adenosine release can be increased by supplying substrates for the PSP (d-ribose and adenine), we now explore the expression of DNPB and PSP enzymes in hippocampal neurons and astrocytes based on available transcriptomic data. We find that key enzymes of the PSP are expressed at higher levels than those in the DNPB pathway, and that PSP enzymes are expressed at higher levels in neurons than in astrocytes. These data reflect the importance of the PSP in the mammalian brain and imply that pharmacological targeting of the PSP may be particularly beneficial to neurons at times of metabolic stress. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.
Topics: Animals; Purines; Adenosine; Brain Injuries; Mammals; Adenosine Triphosphate; Energy Metabolism; Purine Nucleotides
PubMed: 36493858
DOI: 10.1016/j.neuropharm.2022.109370 -
Journal of Visualized Experiments : JoVE Nov 2021Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide...
Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.
Topics: Coenzymes; Flavin-Adenine Dinucleotide; NAD; NADP; Optical Imaging
PubMed: 34842243
DOI: 10.3791/63282 -
MAbs 2022Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine...
Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies. 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund's complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4',6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund's adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter.
Topics: Adenosine Diphosphate Ribose; Animals; CHO Cells; Cricetinae; Cricetulus; Cyclic ADP-Ribose; Humans; Immunoglobulin G; Leukocytes, Mononuclear; NAD; NADP; Niacinamide; Nicotinamide Mononucleotide; Ribose; Sirtuins
PubMed: 35867844
DOI: 10.1080/19420862.2022.2095949 -
Molecular Cancer Research : MCR Jan 2024Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is...
UNLABELLED
Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis.
IMPLICATIONS
SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.
Topics: Humans; Small Cell Lung Carcinoma; Purines; Purine Nucleotides; Hypoxanthine Phosphoribosyltransferase; Lung Neoplasms
PubMed: 37773022
DOI: 10.1158/1541-7786.MCR-23-0386 -
Annual Review of Genetics Nov 2021Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp... (Review)
Review
Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp and ppGpp, collectively (p)ppGpp, have well-documented regulatory roles in gene expression and protein translation. Recent work has highlighted another key function of (p)ppGpp: inducing rapid and coordinated changes in cellular metabolism by regulating enzymatic activities, especially those involved in purine nucleotide synthesis. Failure of metabolic regulation by (p)ppGpp results in the loss of coordination between metabolic and macromolecular processes, leading to cellular toxicity. In this review, we document how (p)ppGpp and newly characterized nucleotides pGpp and (p)ppApp directly regulate these enzymatic targets for metabolic remodeling. We examine targets' common determinants for alarmone interaction as well as their evolutionary diversification. We highlight classical and emerging themes in nucleotide signaling, including oligomerization and allostery along with metabolic interconversion and crosstalk, illustrating how they allow optimized bacterial adaptation to their environmental niches.
Topics: Bacteria; Bacterial Proteins; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Nucleotides
PubMed: 34416118
DOI: 10.1146/annurev-genet-021821-025827 -
Journal of Pharmacological Sciences Oct 2022Adenosine triphosphate (ATP) initially attracted attention as a neurotransmitter, with much research conducted on the regulation of neurotransmission in the autonomic... (Review)
Review
Adenosine triphosphate (ATP) initially attracted attention as a neurotransmitter, with much research conducted on the regulation of neurotransmission in the autonomic and central nervous systems. ATP is also abundant as an energy currency in all living cells and is released into extracellular spaces by various regulated mechanisms. The role of ATP and related purine and pyrimidine nucleotides as extracellular signaling molecules in the regulation of immune cell functions has been reported as evidence for purinergic signaling and has become the focus of attention as therapeutic targets for various diseases. Mast cells (MCs) are distributed in tissues in contact with the outside environment and are the first immune cells to respond to non-microbial environmental antigens. Although extracellular ATP is known as an activator of MCs, the details remain to be investigated. Based on our series of studies, this review describes the unique features of ionotropic P2X4 receptor signals in MC functions. The role of purinergic signaling may exist in combination with various physiological, chemical and physical stimuli. The characteristics of P2X4 receptor-mediated action in MCs described in this article may provide clues to reveal the previously unknown effects induced by purinergic signaling.
Topics: Adenosine Triphosphate; Humans; Hypersensitivity; Mast Cells; Receptors, Purinergic P2X4; Signal Transduction
PubMed: 36055757
DOI: 10.1016/j.jphs.2022.07.005 -
Cells May 2021Alzheimer's disease (AD) is a widespread neurodegenerative pathology responsible for about 70% of all cases of dementia. Adenosine is an endogenous nucleoside that... (Review)
Review
Alzheimer's disease (AD) is a widespread neurodegenerative pathology responsible for about 70% of all cases of dementia. Adenosine is an endogenous nucleoside that affects neurodegeneration by activating four membrane G protein-coupled receptor subtypes, namely P1 receptors. One of them, the A subtype, is particularly expressed in the brain at the striatal and hippocampal levels and appears as the most promising target to counteract neurological damage and adenosine-dependent neuroinflammation. Extracellular nucleotides (ATP, ADP, UTP, UDP, etc.) are also released from the cell or are synthesized extracellularly. They activate P2X and P2Y membrane receptors, eliciting a variety of physiological but also pathological responses. Among the latter, the chronic inflammation underlying AD is mainly caused by the P2X7 receptor subtype. In this review we offer an overview of the scientific evidence linking P1 and P2 mediated purinergic signaling to AD development. We will also discuss potential strategies to exploit this knowledge for drug development.
Topics: Alzheimer Disease; Animals; Humans; Inflammation; Purine Nucleotides; Receptors, Purinergic
PubMed: 34065393
DOI: 10.3390/cells10051267 -
Molecular Psychiatry Sep 2021Phosphodiesterases (PDEs) are enzymes involved in the homeostasis of both cAMP and cGMP. They are members of a family of proteins that includes 11 subfamilies with... (Review)
Review
Phosphodiesterases (PDEs) are enzymes involved in the homeostasis of both cAMP and cGMP. They are members of a family of proteins that includes 11 subfamilies with different substrate specificities. Their main function is to catalyze the hydrolysis of cAMP, cGMP, or both. cAMP and cGMP are two key second messengers that modulate a wide array of intracellular processes and neurobehavioral functions, including memory and cognition. Even if these enzymes are present in all tissues, we focused on those PDEs that are expressed in the brain. We took into consideration genetic variants in patients affected by neurodevelopmental disorders, phenotypes of animal models, and pharmacological effects of PDE inhibitors, a class of drugs in rapid evolution and increasing application to brain disorders. Collectively, these data indicate the potential of PDE modulators to treat neurodevelopmental diseases characterized by learning and memory impairment, alteration of behaviors associated with depression, and deficits in social interaction. Indeed, clinical trials are in progress to treat patients with Alzheimer's disease, schizophrenia, depression, and autism spectrum disorders. Among the most recent results, the application of some PDE inhibitors (PDE2A, PDE3, PDE4/4D, and PDE10A) to treat neurodevelopmental diseases, including autism spectrum disorders and intellectual disability, is a significant advance, since no specific therapies are available for these disorders that have a large prevalence. In addition, to highlight the role of several PDEs in normal and pathological neurodevelopment, we focused here on the deregulation of cAMP and/or cGMP in Down Syndrome, Fragile X Syndrome, Rett Syndrome, and intellectual disability associated with the CC2D1A gene.
Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Humans; Neurodevelopmental Disorders; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases
PubMed: 33414502
DOI: 10.1038/s41380-020-00997-9 -
International Journal of Molecular... Jun 2020A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone...
A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPβS) in a 1-1000 μM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100-1000 μM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 μM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.
Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Affinity Labels; Apoptosis; Cell Cycle; Cell Movement; Cell Proliferation; Extracellular Matrix; Humans; Leukemia, Myeloid, Acute; Thionucleotides; Tumor Cells, Cultured
PubMed: 32580317
DOI: 10.3390/ijms21124425 -
The FEBS Journal Dec 2022ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA...
ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.
Topics: ADP Ribose Transferases; Protein Biosynthesis; Adenosine Diphosphate Ribose; Adenosine Diphosphate
PubMed: 34323016
DOI: 10.1111/febs.16142