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The Biochemical Journal Jan 2020PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has...
PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.
Topics: Acidobacteria; Adenosine Diphosphate; Allosteric Regulation; Bacterial Proteins; Cyclic GMP; Models, Molecular; Oxidoreductases; Protein Binding; Protein Domains
PubMed: 31868878
DOI: 10.1042/BCJ20190809 -
Seminars in Cell & Developmental Biology Dec 2011Although all myosin motors follow the same basic cross-bridge cycle, they display a large variety in the rates of transition between different states in the cycle,... (Review)
Review
Although all myosin motors follow the same basic cross-bridge cycle, they display a large variety in the rates of transition between different states in the cycle, allowing each myosin to be finely tuned for a specific task. Traditionally, myosins have been classified by sequence analysis into a large number of sub-families (∼35). Here we use a different method to classify the myosin family members which is based on biochemical and mechanical properties. The key properties that define the type of mechanical activity of the motor are duty ratio (defined as the fraction of the time myosin remains attached to actin during each cycle), thermodynamic coupling of actin and nucleotide binding to myosin and the degree of strain-sensitivity of the ADP release step. Based on these properties we propose to classify myosins into four different groups: (I) fast movers, (II) slow/efficient force holders, (III) strain sensors and (IV) gates.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Humans; Kinetics; Myosins; Thermodynamics
PubMed: 22001381
DOI: 10.1016/j.semcdb.2011.09.015 -
Platelets Nov 2022Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively....
Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.
Topics: Adenosine Diphosphate; Adenylyl Cyclases; Blood Platelets; Calcium; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Thionucleotides; Type C Phospholipases; rap GTP-Binding Proteins
PubMed: 35514261
DOI: 10.1080/09537104.2022.2071855 -
Investigative Ophthalmology & Visual... Oct 2020Adenosine triphosphate (ATP) is involved in the diameter regulation of retinal vessels. The compound has been shown to induce both constriction and dilatation, but the... (Comparative Study)
Comparative Study
PURPOSE
Adenosine triphosphate (ATP) is involved in the diameter regulation of retinal vessels. The compound has been shown to induce both constriction and dilatation, but the detailed mechanisms underlying these effects and the site of action of the compound are not known in detail. Therefore, the purpose of the present study was to investigate whether the vasoactive effects of ATP on retinal vessels depend on intra- and extravascular application, and to study whether the effects differ at different vascular branching levels.
METHODS
Diameter changes in arterioles, pre-capillary arterioles, and capillaries were studied in perfused porcine hemiretinas (n = 48) ex vivo after intra- and extravascular application of the nondegradable ATP analogue ATP-γ-S or ATP in the presence or not of antagonists to the CD73/ecto-5'-nucleotidase (AOPCP), the P2-purinergic receptor (PPADS), the A3-adenosine receptor (MRS1523), and the synthesis of cyclooxygenase products (ibuprofen).
RESULTS
Intravascular ATP-induced constriction and extravascular ATP-induced dilatation of retinal arterioles, pre-capillary arterioles and capillaries, and dilatation was inhibited by ibuprofen. Both constriction and dilatation of arterioles were inhibited by antagonizing ATP degradation. Furthermore, constriction at all three branching levels was antagonized by blocking the A3 purinoceptor, whereas constriction in arterioles and pre-capillary arterioles was antagonized by blocking the P2 purinoceptor.
CONCLUSIONS
ATP affects the diameter of retinal arterioles, pre-capillary arterioles, and capillaries through different pathways, and the effects depend on whether the compound is administered intravascularly or extravascularly. This may form the basis for selective interventions on retinal vascular disease with differential involvement of vessels at different branching levels.
Topics: 5'-Nucleotidase; Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Animals; Arterioles; Cyclooxygenase Inhibitors; Endovascular Procedures; Enzyme Inhibitors; Ibuprofen; Platelet Aggregation Inhibitors; Retinal Artery; Sus scrofa; Vasoconstriction; Vasodilation
PubMed: 33035289
DOI: 10.1167/iovs.61.12.8 -
Angewandte Chemie (International Ed. in... Jan 2024The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as...
The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.
Topics: Histidine; Solid-Phase Synthesis Techniques; Peptides; ADP-Ribosylation; Adenosine Diphosphate; Adenosine Diphosphate Ribose
PubMed: 37903139
DOI: 10.1002/anie.202313317 -
The Journal of Biological Chemistry Sep 1986The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic...
The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Calorimetry; Mathematics; Myosin Subfragments; Osmolar Concentration; Thermodynamics
PubMed: 3528161
DOI: No ID Found -
Acta Crystallographica. Section F,... Jun 2018Nucleoside diphosphate kinases (NDKs) are implicated in a wide variety of cellular functions owing to their enzymatic conversion of NDP to NTP. NDK from Borrelia...
Nucleoside diphosphate kinases (NDKs) are implicated in a wide variety of cellular functions owing to their enzymatic conversion of NDP to NTP. NDK from Borrelia burgdorferi (BbNDK) was selected for functional and structural analysis to determine whether its activity is required for infection and to assess its potential for therapeutic inhibition. The Seattle Structural Genomics Center for Infectious Diseases (SSGCID) expressed recombinant BbNDK protein. The protein was crystallized and structures were solved of both the apoenzyme and a liganded form with ADP and vanadate ligands. This provided two structures and allowed the elucidation of changes between the apo and ligand-bound enzymes. Infectivity studies with ndk transposon mutants demonstrated that NDK function was important for establishing a robust infection in mice, and provided a rationale for therapeutic targeting of BbNDK. The protein structure was compared with other NDK structures found in the Protein Data Bank and was found to have similar primary, secondary, tertiary and quaternary structures, with conserved residues acting as the catalytic pocket, primarily using His132 as the phosphohistidine-transfer residue. Vanadate and ADP complexes model the transition state of this phosphoryl-transfer reaction, demonstrating that the pocket closes when bound to ADP, while allowing the addition or removal of a γ-phosphate. This analysis provides a framework for the design of potential therapeutics targeting BbNDK inhibition.
Topics: Adenosine Diphosphate; Amino Acid Sequence; Animals; Binding Sites; Borrelia burgdorferi; Female; Mice; Mice, Inbred C3H; Nucleoside-Diphosphate Kinase; Protein Structure, Secondary; Protein Structure, Tertiary; Vanadates
PubMed: 29870023
DOI: 10.1107/S2053230X18007392 -
Biochemical and Biophysical Research... Sep 2018The decapping exoribonuclease DXO functions in pre-mRNA capping quality control, and shows multiple biochemical activities such as decapping, deNADding,...
The decapping exoribonuclease DXO functions in pre-mRNA capping quality control, and shows multiple biochemical activities such as decapping, deNADding, pyrophosphohydrolase, and 5'-3' exoribonuclease activities. Previous studies revealed the molecular mechanisms of DXO based on the structures in complexes with a product, substrate mimic, cap analogue, and 3'-NADP. Despite several reports on the substrate-specific reaction mechanism, the inhibitory mechanism of DXO remains elusive. Here, we demonstrate that adenosine 3', 5'-bisphosphate (pAp), a known inhibitor of the 5'-3' exoribonuclease Xrn1, inhibits the nuclease activity of DXO based on the results of structural and biochemical experiments. We determined the crystal structure of the DXO-pAp-Mg complex at 1.8 Å resolution. In comparison with the DXO-RNA product complex, the position of pAp is well superimposed with the first nucleotide of the product RNA in the vicinity of two magnesium ions. Furthermore, biochemical assays showed that the inhibition by pAp is comparable between Xrn1 and DXO. Collectively, these structural and biochemical studies reveal that pAp inhibits the activities of DXO by occupying the active site to act as a competitive inhibitor.
Topics: Adenosine Diphosphate; Binding Sites; Endoribonucleases; Exoribonucleases; Magnesium; Models, Molecular
PubMed: 30180947
DOI: 10.1016/j.bbrc.2018.08.135 -
International Journal of Molecular... May 2020Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane...
Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6 platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6 platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate G-coupled 5HT and G-coupled adrenergic receptors, respectively, was not affected in GRK6 platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6 platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y, P2Y, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6 platelets. Finally, GRK6 mice exhibited an enhanced and stable thrombus formation after FeCl injury to the carotid artery and shorter tail bleeding times, indicating that GRK6 mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.
Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Blood Platelets; Female; G-Protein-Coupled Receptor Kinases; Gene Expression Regulation; Hemostatics; Male; Mice; Mice, Knockout; Oligopeptides; Phosphorylation; Platelet Activation; Platelet Aggregation; Receptors, G-Protein-Coupled; Thionucleotides; Thrombin; Thromboxane A2
PubMed: 32486261
DOI: 10.3390/ijms21113932 -
Blood Jan 2017
Topics: Adenosine Diphosphate
PubMed: 28082287
DOI: 10.1182/blood-2016-11-751917