-
International Journal of Molecular... Jun 2022AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed...
AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D--Ins(2,3,4,5)P and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.
Topics: 6-Phytase; Acid Phosphatase; Animals; Dinucleoside Phosphates; Escherichia coli; Escherichia coli Proteins; Phytic Acid; Prospective Studies; Protons
PubMed: 35683026
DOI: 10.3390/ijms23116346 -
Journal of Chemical Theory and... Jun 2022RNA modulation via small molecules is a novel approach in pharmacotherapies, where the determination of the structural properties of RNA motifs is considered a promising...
RNA modulation via small molecules is a novel approach in pharmacotherapies, where the determination of the structural properties of RNA motifs is considered a promising way to develop drugs capable of targeting RNA structures to control diseases. However, due to the complexity and dynamic nature of RNA molecules, the determination of RNA structures using experimental approaches is not always feasible, and computational models employing force fields can provide important insight. The quality of the force field will determine how well the predictions are compared to experimental observables. Stacking in nucleic acids is one such structural property, originating mainly from London dispersion forces, which are quantum mechanical and are included in molecular mechanics force fields through nonbonded interactions. Geometric descriptions are utilized to decide if two residues are stacked and hence to calculate the stacking free energies for RNA dinucleoside monophosphates (DNMPs) through statistical mechanics for comparison with experimental thermodynamics data. Here, we benchmark four different stacking definitions using molecular dynamics (MD) trajectories for 16 RNA DNMPs produced by two different force fields (RNA-IL and ff99OL3) and show that our stacking definition better correlates with the experimental thermodynamics data. While predictions within an accuracy of 0.2 kcal/mol at 300 K were observed in RNA CC, CU, UC, AG, GA, and GG, stacked states of purine-pyrimidine and pyrimidine-purine DNMPs, respectively, were typically underpredicted and overpredicted. Additionally, population distributions of RNA UU DNMPs were poorly predicted by both force fields, implying a requirement for further force field revisions. We further discuss the differences predicted by each RNA force field. Finally, we show that discrete path sampling (DPS) calculations can provide valuable information and complement the MD simulations. We propose the use of experimental thermodynamics data for RNA DNMPs as benchmarks for testing RNA force fields.
Topics: DNA; Dinucleoside Phosphates; Molecular Dynamics Simulation; Nucleic Acid Conformation; Purines; Pyrimidines; RNA; Thermodynamics
PubMed: 35652685
DOI: 10.1021/acs.jctc.2c00178 -
BMC Cardiovascular Disorders May 2022The prevalence and burden of coronary heart disease (CHD) has increased substantially in India, accompanied with increasing need for percutaneous coronary interventions...
BACKGROUND
The prevalence and burden of coronary heart disease (CHD) has increased substantially in India, accompanied with increasing need for percutaneous coronary interventions (PCI). Although a large government-funded insurance scheme in Maharashtra, India covered the cost of PCI for low-income patients, the high cost of post-PCI treatment, especially Dual Antiplatelet Therapy (DAPT), still caused many patients to prematurely discontinue the secondary prevention. Our study aimed to investigate the effectiveness of DAPT adherence on all-cause mortality among post-PCI patients and explore the potential determinants of DAPT adherence in India.
METHOD
We collected clinical data of 4,595 patients undergoing PCI in 110 participating medical centers in Maharashtra, India from 2012 to 2015 by electronic medical records. We surveyed 2527 adult patients who were under the insurance scheme by telephone interview, usually between 6 to 12 months after their revascularization. Patients reporting DAPT continuation in the telephone survey were categorized as DAPT adherence. The outcome of the interest was all-cause mortality within 1 year after the index procedure. Multivariate Cox proportional hazard (PH) model with adjustment of potential confounders and standardization were used to explore the effects of DAPT adherence on all-cause mortality. We further used a multivariate logistic model to investigate the potential determinants of DAPT adherence.
RESULTS
Out of the 2527 patients interviewed, 2064 patients were included in the analysis, of whom 470 (22.8%) discontinued DAPT prematurely within a year. After adjustment for baseline confounders, DAPT adherence was associated with lower one-year all-cause mortality compared to premature discontinuation (less than 6-month), with an adjusted hazard ratio (HR) of 0.52 (95% Confidence Interval (CI) = (0.36, 0.67)). We also found younger patients (OR per year was 0.99 (0.97, 1.00)) and male (vs. female, OR of 1.30 (0.99, 1.70)) had higher adherence to DAPT at one year as did patients taking antihypertensive medications (vs. non medication, OR of 1.57 (1.25, 1.95)).
CONCLUSION
These findings suggest the protective effects of DAPT adherence on 1-year mortality among post-PCI patients in a low-income setting and indicate younger age, male sex and use of other preventive treatments were predictors of higher DAPT adherence.
Topics: Adult; Dinucleoside Phosphates; Drug Therapy, Combination; Female; Humans; India; Male; Percutaneous Coronary Intervention; Platelet Aggregation Inhibitors; Treatment Outcome
PubMed: 35606724
DOI: 10.1186/s12872-022-02677-8 -
Biophysical Journal Jun 2022Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts...
Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant K for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Flavins; Flavoproteins; Gene Expression Regulation, Bacterial; Heme; Repressor Proteins; Rhodobacter sphaeroides
PubMed: 35488435
DOI: 10.1016/j.bpj.2022.04.031 -
Angewandte Chemie (International Ed. in... Jul 2022The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used...
The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion.
Topics: Dinucleoside Phosphates; Mass Spectrometry; Nucleotides; RNA
PubMed: 35445525
DOI: 10.1002/anie.202203067 -
Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.Cell Host & Microbe Jul 2022Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes...
Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.
Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Folic Acid Antagonists; Inflammation; Mice; Staphylococcal Infections; Staphylococcus aureus; Thymidine
PubMed: 35439435
DOI: 10.1016/j.chom.2022.03.028 -
Nature Apr 2022Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria. STING-mediated immunity could be exploited in the...
Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53), promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.
Topics: Cell Cycle Proteins; Cryoelectron Microscopy; Dinucleoside Phosphates; Humans; Immunity, Innate; Ligands; Membrane Proteins; Nucleotides, Cyclic; Tumor Suppressor Proteins
PubMed: 35388221
DOI: 10.1038/s41586-022-04559-7 -
The New Phytologist Jun 2022The amount of inorganic carbon (C ) fluctuates in aquatic environments. Cyanobacteria evolved a C -concentrating mechanism (CCM) that is regulated at different levels....
The amount of inorganic carbon (C ) fluctuates in aquatic environments. Cyanobacteria evolved a C -concentrating mechanism (CCM) that is regulated at different levels. The regulator SbtB binds to the second messengers cAMP or c-di-AMP and is involved in acclimation to low C (LC) in Synechocystis sp. PCC 6803. Here, we investigated the role of SbtB and of associated second messengers at different C conditions. The transcriptome of wild-type (WT) Synechocystis and the ΔsbtB mutant were compared with Δcya1, a mutant defective in cAMP production, and ΔdacA, a mutant defective in generating c-di-AMP. A defined subset of LC-regulated genes in the WT was already changed in ΔsbtB under high C (HC) conditions. This response of ΔsbtB correlated with a diminished induction of many CCM-associated genes after LC shift in this mutant. The Δcya1 mutant showed less deviation from WT, whereas ΔdacA induced CCM-associated genes under HC. Metabolome analysis also revealed differences between the strains, whereby ΔsbtB showed slower accumulation of 2-phosphoglycolate and ΔdacA differences among amino acids compared to WT. Collectively, these results indicate that SbtB regulates a subset of LC acclimation genes while c-di-AMP and especially cAMP appear to have a lesser impact on gene expression under different C availabilities.
Topics: Bacterial Proteins; Carbon; Carbon Dioxide; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Photosynthesis; Second Messenger Systems; Synechocystis; Transcriptome
PubMed: 35285042
DOI: 10.1111/nph.18094 -
Chembiochem : a European Journal of... Apr 2022The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the...
The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-c GAMP and 3'3'-c GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-c GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-c GAMP in THP-1 cells.
Topics: Animals; DNA; Dinucleoside Phosphates; Humans; Interferons; Mammals; Membrane Proteins; Mice; Nucleotidyltransferases; Second Messenger Systems
PubMed: 35189023
DOI: 10.1002/cbic.202200005 -
Nature Communications Feb 2022In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed...
In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.
Topics: Animals; BCG Vaccine; CD8-Positive T-Lymphocytes; Cancer Vaccines; Cell Line, Tumor; Cytokines; Dinucleoside Phosphates; Humans; Immunity, Innate; Macrophages; Membrane Proteins; Mice; Myeloid Cells; Pathogen-Associated Molecular Pattern Molecules; Rats; Urinary Bladder Neoplasms; Urothelium; Vaccination
PubMed: 35169141
DOI: 10.1038/s41467-022-28509-z