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Enzyme and Microbial Technology Feb 2021Cyclic di-AMP is a bacterial nucleotide second messenger and evaluated as a potential vaccine adjuvant candidate. Here, we report a practical and economical enzymatic...
Cyclic di-AMP is a bacterial nucleotide second messenger and evaluated as a potential vaccine adjuvant candidate. Here, we report a practical and economical enzymatic method for gram-scale preparation of c-di-AMP using an immobilized Vibrio cholerae dinucleotide cyclase DncV. The method mainly includes four steps: preparation of DncV-immobilized resin, enzymatic synthesis of c-di-AMP, purification using macroporous absorption resin SP207, and desiccation using rotary evaporation and lyophilization. Enzymatic synthesis is the most critical step, and almost all substrate ATP was converted to c-di-AMP under an optimum condition in which 300 mL of 300 mM NHAc/NH pH 9.5 buffer supplemented with 20 mM MnCl, 10 mM ATP and 4 mL of DncV-immobilized resin containing ∼19 mg DncV were incubated at 30 °C overnight. After purification, up to 1 g of the diammonium salt of c-di-AMP with weight purity of ≥98% was obtained as white powder, which corresponds to an overall yield of ∼80% based on the ATP input into the reaction. The method is easily performed in laboratory to prepare c-di-AMP on a gram scale and could be used in industry on a large scale.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Vibrio cholerae
PubMed: 33375968
DOI: 10.1016/j.enzmictec.2020.109700 -
Journal of Virology Sep 2021Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate...
Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a nearinfrared fluorescent protein (iRFP) upstream of . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication and reporter expression and . Both codon-optimized reporter viruses could be visualized during coinfection and reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.
Topics: Animals; CD4-Positive T-Lymphocytes; Dinucleoside Phosphates; Gene Expression; Genes, Reporter; HIV Infections; HIV-1; Humans; Luciferases; Luminescent Measurements; Luminescent Proteins; Mice; Optical Imaging; Viremia; Virus Replication; Whole Body Imaging
PubMed: 34232063
DOI: 10.1128/JVI.00449-21 -
The Journal of Biological Chemistry Jul 2022The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and...
The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.
Topics: Adenine; Adenosine Monophosphate; Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates
PubMed: 35714772
DOI: 10.1016/j.jbc.2022.102144 -
Nucleic Acids Research Sep 2021The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with...
The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.
Topics: ATP-Binding Cassette Transporters; Biological Transport; Crystallography, X-Ray; DNA, Bacterial; DNA-Binding Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Potassium; Protein Domains; Second Messenger Systems; Streptococcus agalactiae; Transcription Factors
PubMed: 34432045
DOI: 10.1093/nar/gkab736 -
Current Protocols Jan 2021This article describes a reliable and efficient method for synthesis of the dinucleotide cap analog m G[5']ppp[5']G containing a locked nucleic acid moiety. The required...
This article describes a reliable and efficient method for synthesis of the dinucleotide cap analog m G[5']ppp[5']G containing a locked nucleic acid moiety. The required LNA intermediate for the final coupling reaction, m GDP, is prepared in six steps starting from 5'-DMTr-N-DMF LNA guanosine. The overall reaction involves removal of DMTr and DMF groups, 5' monophosphorylation, imidazolide formation, diphosphorylation, and regioselective m methylation. The final coupling reaction of m GDP with ImGMP in the presence of zinc chloride as a catalyst affords m G[5']ppp[5']G in 59% yield. © 2021 Wiley Periodicals LLC. Basic Protocol: Synthesis of an LNA-substituted dinucleotide cap analog Support Protocol: Preparation of the tris(tributylammonium) phosphate linker.
Topics: Dinucleoside Phosphates; Guanosine; Methylation; Oligonucleotides
PubMed: 33484497
DOI: 10.1002/cpz1.22 -
Plant Physiology and Biochemistry : PPB Feb 2020It is known that the concentration of dinucleoside polyphosphates (NpN's) in cells increases under stress and that adverse environmental factors induce biosynthesis of...
It is known that the concentration of dinucleoside polyphosphates (NpN's) in cells increases under stress and that adverse environmental factors induce biosynthesis of phenylpropanoids, which protect the plant against stress. Previously, we showed that purine NpN's such as ApA and ApA induce both the activity of enzymes of the phenylpropanoid pathway and the expression of relevant genes in Arabidopsis seedlings. Moreover, we showed that ApA induced stilbene biosynthesis in Vitis vinifera cv. Monastrell suspension cultured cells. Data presented in this paper show that pyrimidine-containing NpN's also modify the biosynthesis of stilbenes, affecting the transcript level of genes encoding key enzymes of the phenylpropanoid pathway and of these, UpU caused the most effective accumulation of trans-resveratrol in the culture media. Similar effect was caused by ApA and GpG. Other pyrimidine NpN's, such as CpC, CpC, and ApC, strongly inhibited the biosynthesis of stilbenes, but markedly (6- to 8-fold) induced the expression of the cinnamoyl-CoA reductase gene that controls lignin biosynthesis. Purine counterparts also clearly induced biosynthesis of trans-resveratrol and trans-piceid, but only slightly induced the expression of genes involved in lignin biosynthesis. In cells, UpU caused a greater accumulation of trans-resveratrol and trans-piceid than did UpU. Each of the NpN's studied induced expression of the gene encoding the resveratrol transporter VvABCG44, which operates within the Vitis vinifera cell membrane. AMP, GMP, UMP, and CMP, potential products of NpN degradation, did not affect the accumulation of stilbenes. The results obtained strongly support that NpN's play a role as signaling molecules in plants.
Topics: Cells, Cultured; Dinucleoside Phosphates; Gene Expression Regulation, Plant; Purines; Pyrimidines; Stilbenes; Vitis
PubMed: 31855818
DOI: 10.1016/j.plaphy.2019.12.015 -
Apoptosis : An International Journal on... Jun 2021Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of...
Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negative breast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1 cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.
Topics: Apoptosis; Cell Death; Cell Line, Tumor; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Membrane Proteins; Mitochondria; Protein Binding; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Triple Negative Breast Neoplasms
PubMed: 33840002
DOI: 10.1007/s10495-021-01669-x -
Stresses that Raise NpA Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.Molecular Cell Sep 2019Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has...
Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has been delineated in eukaryotes, and no receptor that mediates the influence of NpNs has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular NpN concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated NpN concentrations in bacteria may result from widespread Np capping, leading to altered RNA stability and consequent changes in gene expression.
Topics: Acid Anhydride Hydrolases; Dinucleoside Phosphates; Escherichia coli K12; Escherichia coli Proteins; RNA Stability; RNA, Bacterial
PubMed: 31178354
DOI: 10.1016/j.molcel.2019.05.031 -
Angewandte Chemie (International Ed. in... Feb 2020The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective...
The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA-based ArMs containing duplex and G-quadruplex scaffolds have been widely investigated, yet RNA-based ArMs are scarce. Here we report that a cyclic dinucleotide of c-di-AMP and Cu ions assemble into an artificial metalloribozyme (c-di-AMP⋅Cu ) that enables catalysis of enantioselective Friedel-Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c-di-AMP⋅Cu gives rise to a 20-fold rate acceleration compared to Cu ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c-di-AMP⋅Cu metalloribozyme is suggested in which two c-di-AMP form a dimer scaffold and the Cu ion is located in the center of an adenine-adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.
Topics: Catalysis; Copper; Cycloaddition Reaction; Density Functional Theory; Dimerization; Dinucleoside Phosphates; G-Quadruplexes; Kinetics; Metalloproteins; Stereoisomerism; Substrate Specificity; Water
PubMed: 31825550
DOI: 10.1002/anie.201912962 -
Biochimica Et Biophysica Acta. General... Nov 2021Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional...
Biochemical, crystallographic and biophysical characterization of histidine triad nucleotide-binding protein 2 with different ligands including a non-hydrolyzable analog of Ap4A.
BACKGROUND
Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.
METHODS
An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.
RESULTS
HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.
CONCLUSIONS
Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.
SIGNIFICANCE
Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.
Topics: Dinucleoside Phosphates; Humans; Ligands; Mitochondrial Proteins
PubMed: 34329705
DOI: 10.1016/j.bbagen.2021.129968