-
The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.The FEBS Journal Jun 2023Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly...
Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.
Topics: Carrier Proteins; Streptococcus agalactiae; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Potassium
PubMed: 36629470
DOI: 10.1111/febs.16724 -
International Journal of Molecular... Dec 2022Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated...
Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.
Topics: Nucleosides; Phosphates; Hydrolysis; Diphosphates; Chalcogens
PubMed: 36555224
DOI: 10.3390/ijms232415582 -
Angewandte Chemie (International Ed. in... Feb 2023Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling...
Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Ap As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap A levels during cellular stress differs from when Ap A is present at low concentrations, allowing response to extracellular cues.
Topics: Humans; Guanosine Pentaphosphate; Proteins; Adenosine Monophosphate; Dinucleoside Phosphates; Protein Processing, Post-Translational
PubMed: 36524454
DOI: 10.1002/anie.202213279 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... Nov 2022Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the... (Review)
Review
Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the expression of target proteins, c-di-AMP regulates the physiological functions of bacteria, including maintaining osmotic pressure, balancing central metabolism, monitoring DNA damage, and controlling biofilm and spore formation. As a new pathogen-associated molecular pattern (PAMP), it binds to the host pattern recognition receptor (PRR), induces cyclic GMP-AMP synthase (cGAS)-STING signal axis to produce type Ⅰ interferon by activating the stimulator of interferon genes (STING), and promotes the secretion of inflammatory factors through nuclear factor κB (NF-κB) signaling pathway, thereby playing an important role in host immunity to bacterial infection and tumorigenesis. Due to its immunogenicity, c-di-AMP could be used as an immune adjuvant to provide new targets for the development of vaccines. However, the specific mechanism of action of c-di-AMP in host immunity awaits further exploration. Herein, we presented the structure and biological characteristics of c-di-AMP, and summarized the possible mechanism of c-di-AMP's regulation of host immune response. In addition, we also reported the latest findings on using c-di-AMP as an immune adjuvant in clinical treatment. Research on the function of c-di-AMP and its mechanism of action on host immune response provides new ideas for finding clinical solutions to bacterial resistance, infection control, tumor prevention, and vaccine development in the future.
Topics: Dinucleoside Phosphates; Bacteria; Biofilms; Signal Transduction
PubMed: 36443059
DOI: 10.12182/20220860102 -
RNA (New York, N.Y.) Feb 2023mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such...
mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such therapeutics are an attractive alternative for DNA-based vaccines since they provide material that can be used with no risk of genomic integration. Additionally, mRNA can be quite easily engineered to introduce modifications for different applications or to modulate its properties, for example, to increase translational efficiency or stability, which is not available for DNA vectors. Here, we describe the use of N2 modified dinucleotide cap analogs as components of mRNA transcripts. The compounds obtained showed very promising biological properties while incorporated into mRNA. The presented N2-guanine modifications within the cap structure ensure proper attachment of the dinucleotide to the transcripts in the IVT reaction, guarantees their incorporation only in the correct orientation, and enables highly efficient translation of mRNA both in the in vitro translation system and in human HEK293 cells.
Topics: Humans; RNA, Messenger; Protein Biosynthesis; RNA Cap Analogs; HEK293 Cells; Dinucleoside Phosphates; Vaccines
PubMed: 36418172
DOI: 10.1261/rna.079460.122 -
The Journal of Physical Chemistry. B Dec 2022Autofluorescence imaging has been widely applied as advanced noninvasive diagnostics for in vivo and ex vivo tissues. The optical redox ratio (ORR), which is defined as...
Autofluorescence imaging has been widely applied as advanced noninvasive diagnostics for in vivo and ex vivo tissues. The optical redox ratio (ORR), which is defined as the fluorescence intensity ratio between reduced nicotine adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), has been used as a diagnostic parameter strongly, because NADH and FAD play an important role in energetic and respiratory metabolism as coenzymes. The ORR method has provided successful assessment in cancer diagnosis including breast, cervical, and oral cancer; few studies have been reported about optical and chemical interference between two molecules resulting in a change in ORR values. In this study, we investigated the variations in ORR values of NADH/FAD mixtures dissolved in tris(hydroxymethyl)aminomethane, phosphate buffer, and deionized water environments. In vitro solutions were prepared in various concentration ratios and the experimental and theoretical ORR values were obtained from fluorescence and absorption spectra in time series. Based on the spectroscopic analysis, we concluded that the inner filter effect causes an instant decrease in FAD fluorescence just after dissolution and that the oxidation-reduction coupled with oxygenation reaction results in time-varying decreases in NADH fluorescence and FAD emission.
Topics: Adenine; Nicotine; Flavin-Adenine Dinucleotide; NAD; Dinucleoside Phosphates; Oxidation-Reduction
PubMed: 36399328
DOI: 10.1021/acs.jpcb.2c05292 -
Current Protocols Nov 2022This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pA pA, pA pG,...
This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pA pA, pA pG, and pA pU that utilizes phosphoramidite chemistry. The overall synthetic strategy involves three steps. The first step involves the coupling reaction between 5'-O-MMT protected nucleoside-3'-O-phosphoramidite and a protected nucleoside containing a free 5'-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert-butyl hydroperoxide to give the corresponding protected N pN. Next, the 5'-O-MMT is cleaved under 3% trichloroacetic acid in dichloromethane conditions. Finally, the 5'-hydroxyl group is phosphorylated by the use of an activated bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite using tetrazole, followed by the oxidation of trivalent to pentavalent phosphorus using tert-butyl hydroperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pN pN, in good yields with high purity (>99.5%). © 2022 Wiley Periodicals LLC.
Topics: Dinucleoside Phosphates; Nucleosides; RNA; tert-Butylhydroperoxide; Tetrazoles
PubMed: 36342272
DOI: 10.1002/cpz1.583 -
Nature Dec 2022Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase...
Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A1. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.
Topics: Animals; Humans; Cryoelectron Microscopy; Dinucleoside Phosphates; Folic Acid; Folic Acid Antagonists; Mammals; Nucleotides, Cyclic; Reduced Folate Carrier Protein
PubMed: 36265513
DOI: 10.1038/s41586-022-05452-z -
Journal of Peptide Science : An... Mar 2023Intracellular dinucleoside polyphosphates (Np Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap A) is one of the...
Intracellular dinucleoside polyphosphates (Np Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit.
Topics: Humans; Dinucleoside Phosphates; Neoplasm Proteins; Guanosine Pentaphosphate; Acid Anhydride Hydrolases; HEK293 Cells; Proteomics; Neoplasms
PubMed: 36264037
DOI: 10.1002/psc.3458 -
Frontiers in Immunology 2022Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through...
Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after infection. It was found that rBCG-DisA resided longer than BCG in the lung of infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against infection. Our findings provided experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What's more, rBCG-DisA showed promising potential in prime-boost strategy against infection in adults.
Topics: Acyltransferases; Adenosine Monophosphate; Adjuvants, Immunologic; Adjuvants, Pharmaceutic; Animals; Antigens, Bacterial; BCG Vaccine; Cyclic AMP; Cytokines; Dinucleoside Phosphates; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Mycobacterium tuberculosis; Tuberculosis, Lymph Node; Vaccines, Synthetic
PubMed: 36081510
DOI: 10.3389/fimmu.2022.943667