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Nature Apr 2023Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian...
Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.
Topics: Bacterial Proteins; Circadian Clocks; Circadian Rhythm; Phosphorylation; Rhodobacter sphaeroides; Crystallography, X-Ray; Cryoelectron Microscopy; Adenosine Triphosphate; Adenosine Diphosphate; Kinetics; Protein Folding; Protein Conformation; Allosteric Regulation
PubMed: 36949197
DOI: 10.1038/s41586-023-05836-9 -
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 -
Genes & Development Mar 2020ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life.... (Review)
Review
ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.
Topics: ADP-Ribosylation; Adenosine Diphosphate; Humans; Hydrolases; Protein Domains; Structure-Activity Relationship
PubMed: 32029451
DOI: 10.1101/gad.334631.119 -
Purinergic Signalling Mar 2023Pathogenesis of ischemic stroke is mainly characterized by thrombosis and neuroinflammation. Purinergic signaling pathway constitutes adenosine triphosphate (ATP),... (Review)
Review
Pathogenesis of ischemic stroke is mainly characterized by thrombosis and neuroinflammation. Purinergic signaling pathway constitutes adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO). ATP is hydrolyzed to ADP and then to AMP by extracellular nucleotidase CD39; AMP is subsequently converted to adenosine by CD73. All these nucleotides and nucleosides act on purinergic receptors protecting against thrombosis and inhibit inflammation. In addition, many physical methods have been found to play a neuroprotective role through purinergic signaling. This review mainly introduces the role and potential mechanism of purinergic signalings in the treatment of ischemic stroke, so as to provide reference for seeking new treatment methods for stroke.
Topics: Humans; Ischemic Stroke; Antigens, CD; Adenosine; Adenosine Triphosphate; Signal Transduction; Adenosine Diphosphate; Thrombosis; Adenosine Monophosphate; 5'-Nucleotidase; Apyrase
PubMed: 36370253
DOI: 10.1007/s11302-022-09905-y -
Redox Biology Nov 2021Purinergic signaling is a cell communication pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides are released in physiological... (Review)
Review
Purinergic signaling is a cell communication pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides are released in physiological and pathological circumstances activating purinergic type 2 receptors (P2 receptors): P2X ion channels and P2Y G protein-coupled receptors. The activation of these receptors triggers the production of reactive oxygen and nitrogen species and alters antioxidant defenses, modulating the redox biology of cells. The activation of P2 receptors is controlled by ecto-enzymes named ectonucleotidases, E-NTPDase1/CD39 and ecto-5'-nucleotidase/CD73) being the most relevant. The first enzyme hydrolyzes adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP), and the second catalyzes the hydrolysis of AMP to adenosine. The activity of these enzymes is diminished by oxidative stress. Adenosine actives P1 G-coupled receptors that, in general, promote the maintenance of redox hemostasis by decreasing reactive oxygen species (ROS) production and increase antioxidant enzymes. Intracellular purine metabolism can also contribute to ROS generation via xanthine oxidase activity, which converts hypoxanthine into xanthine, and finally, uric acid. In this review, we describe the mechanisms of redox biology modulated by purinergic signaling and how this signaling may be affected by disturbances in the redox homeostasis of cells.
Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Biology; Oxidation-Reduction
PubMed: 34563872
DOI: 10.1016/j.redox.2021.102137 -
Journal of the American Chemical Society Oct 2023Under enzyme catalysis, adenosine triphosphate (ATP) transfers a phosphoryl group to canonical ribonucleotide diphosphates (NDPs) to form ribonucleotide triphosphates...
Under enzyme catalysis, adenosine triphosphate (ATP) transfers a phosphoryl group to canonical ribonucleotide diphosphates (NDPs) to form ribonucleotide triphosphates (NTPs), the direct biosynthetic precursors to RNA. However, it remains unclear whether the phosphorylation of NDPs could have occurred in water before enzymes existed and why an adenosine derivative, rather than another canonical NTP, typically performs this function. Here, we show that adenosine diphosphate (ADP) in the presence of Fe or Al promotes phosphoryl transfer from acetyl phosphate to all canonical NDPs to produce their corresponding NTP in water at room temperature and in the absence of enzymes. No other NDPs were found to promote phosphorylation, giving insight into why adenosine derivatives specifically became used for this purpose in biology. The metal-ADP complexes also promote phosphoryl transfer to ribonucleoside monophosphates (NMPs) to form a mixture of the corresponding NDPs and NTPs, albeit less efficiently. This work represents a rare example in which a single nucleotide carries out a function critical to biology without enzymes. ADP-metal complexes may have played an important role in nucleotide phosphorylation in prebiotic chemistry.
Topics: Ribonucleotides; Phosphorylation; Coordination Complexes; Adenosine Triphosphate; Adenosine Diphosphate; Adenosine; Water
PubMed: 37750669
DOI: 10.1021/jacs.3c08047 -
The Biochemical Journal Jun 2022Infection with schistosomes (blood flukes) can result in the debilitating disease schistosomiasis. These parasites survive in their host for many years, and we...
Infection with schistosomes (blood flukes) can result in the debilitating disease schistosomiasis. These parasites survive in their host for many years, and we hypothesize that proteins on their tegumental surface, interacting with the host microenvironment, facilitate longevity. One such ectoenzyme - the nucleotide pyrophosphatase/phosphodiesterase SmNPP5 can cleave ADP (to prevent platelet aggregation) and NAD (likely preventing Treg apoptosis). A second tegumental ectoenzyme, the glycohydrolase SmNACE, also catabolizes NAD. Here, we undertake a comparative biochemical characterization of these parasite ectoenzymes. Both are GPI-linked and exhibit different optimal pH ranges. While SmNPP5 requires divalent cations, SmNACE does not. The KM values of the two enzymes for NAD at physiological pH differ: SmNPP5, KM = 340 µM ± 44; SmNACE, KM = 49 µM ± 4. NAD cleavage by each enzyme yields different products. SmNPP5 cleaves NAD to form nicotinamide mononucleotide (NMN) and AMP, whereas SmNACE cleaves NAD to generate nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Each enzyme can process the other's reaction product. Thus, SmNACE cleaves NMN (to yield NAM and ribose phosphate) and SmNPP5 cleaves ADPR (yielding AMP and ribose phosphate). Metabolomic analysis of plasma containing adult worms supports the idea that these cleavage pathways are active in vivo. We hypothesize that a primary function of SmNPP5 is to cleave NAD to control host immune cell function and a primary function of SmNACE is to cleave NMN to generate the vital nutrient nicotinamide (vitamin B3) for convenient uptake by the worms. Chemical inhibition of one or both ectoenzymes could upset worm metabolism and control schistosome infection.
Topics: Adenosine Diphosphate Ribose; Adenosine Monophosphate; Animals; NAD; Niacinamide; Schistosoma mansoni
PubMed: 35593185
DOI: 10.1042/BCJ20210784 -
Molecular Cell May 2023PARPs catalyze ADP-ribosylation-a post-translational modification that plays crucial roles in biological processes, including DNA repair, transcription, immune... (Review)
Review
PARPs catalyze ADP-ribosylation-a post-translational modification that plays crucial roles in biological processes, including DNA repair, transcription, immune regulation, and condensate formation. ADP-ribosylation can be added to a wide range of amino acids with varying lengths and chemical structures, making it a complex and diverse modification. Despite this complexity, significant progress has been made in developing chemical biology methods to analyze ADP-ribosylated molecules and their binding proteins on a proteome-wide scale. Additionally, high-throughput assays have been developed to measure the activity of enzymes that add or remove ADP-ribosylation, leading to the development of inhibitors and new avenues for therapy. Real-time monitoring of ADP-ribosylation dynamics can be achieved using genetically encoded reporters, and next-generation detection reagents have improved the precision of immunoassays for specific forms of ADP-ribosylation. Further development and refinement of these tools will continue to advance our understanding of the functions and mechanisms of ADP-ribosylation in health and disease.
Topics: Poly(ADP-ribose) Polymerases; ADP-Ribosylation; Protein Processing, Post-Translational; Adenosine Diphosphate Ribose
PubMed: 37119811
DOI: 10.1016/j.molcel.2023.04.009 -
Photochemical & Photobiological... Jun 2022Zn-salophen complexes are a promising class of fluorescent chemosensors for nucleotides and nucleic acids. We have investigated, by means of steady state UV-Vis,...
Zn-salophen complexes are a promising class of fluorescent chemosensors for nucleotides and nucleic acids. We have investigated, by means of steady state UV-Vis, ultrafast transient absorption, fluorescence emission and time dependent density functional theory (TD-DFT) the behavior of the excited states of a salicylidene tetradentate Schiff base (Sal), its Zn(II) coordination compound (Zn-Sal) and the effect of the interaction between Zn-Sal and adenosine diphosphate (ADP). TD-DFT shows that the deactivation of the excited state of Sal occurs through torsional motion, due to its rotatable bonds and twistable angles. Complexation with Zn(II) causes rigidity so that the geometry changes in the excited states with respect to the ground state structure are minimal. By addition of ADP to a freshly prepared Zn-Sal ethanol solution, a longer relaxation constant, in comparison to Zn-Sal, was measured, indicative of the interaction between Zn-Sal and ADP. After a few days, the Zn-Sal-ADP solution displayed the same static and dynamic behavior of a solution containing only the Sal ligand, demonstrating that the coordination of the ADP anion to Zn(II)leads to the demetallation of the Sal ligand. Fluorescence measurements also revealed an enhanced fluorescence at 375 nm following the addition of ADP to the solution, caused by the presence of 2,3-diamino naphthalene that is formed by demetallation and partial decomposition of the Sal ligand. The efficient fluorescence of this species at 375 nm could be selectively detected and used as a probe for the detection of ADP in solution.
Topics: Adenosine Diphosphate; Ligands; Salicylates; Zinc
PubMed: 35088368
DOI: 10.1007/s43630-021-00165-0 -
Biochemical Society Transactions Jun 2020Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between... (Review)
Review
Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags" the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Allosteric Site; Bile Acids and Salts; Biological Transport; Humans; Kinetics; Lipid Bilayers; Lipids; Membrane Transport Proteins; Molecular Dynamics Simulation; Protein Domains; Protein Folding; Receptors, Somatostatin
PubMed: 32369548
DOI: 10.1042/BST20200290