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Nature Communications Feb 2020It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpNs) and yet their roles and mechanisms of action remain unclear. Here, we show that...
It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpNs serve as RNA caps in Escherichia coli. NpNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpN-caps from RNA. ApaH is able to cleave all NpN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
Topics: Acid Anhydride Hydrolases; DNA-Directed RNA Polymerases; Dinucleoside Phosphates; Escherichia coli; Escherichia coli Proteins; Methylation; Nucleic Acid Conformation; RNA Caps; RNA Stability; RNA, Bacterial
PubMed: 32103016
DOI: 10.1038/s41467-020-14896-8 -
Nature Communications Jan 2022Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense...
Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in β-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.
Topics: Bacteria; Crystallography, X-Ray; Cyclic GMP; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferons; Ligands; Membrane Proteins; Nucleotidyltransferases; Prevotella
PubMed: 35013136
DOI: 10.1038/s41467-021-26583-3 -
Journal of Bacteriology Apr 2023Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to... (Review)
Review
Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.
Topics: Humans; Archaea; Phosphorus-Oxygen Lyases; Bacterial Proteins; Bacteria; Second Messenger Systems; Cyclic AMP; Dinucleoside Phosphates
PubMed: 37022175
DOI: 10.1128/jb.00023-23 -
Microbiology and Molecular Biology... Jun 2024SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and... (Review)
Review
SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and metabolic adaptations, often in response to environmental fluctuations of physical parameters. In the last two decades, cyclic di-AMP has emerged as an important signaling nucleotide in many prokaryotic lineages, including Firmicutes, Actinobacteria, and Cyanobacteria. Its importance is highlighted by the fact that both the lack and overproduction of cyclic di-AMP affect viability of prokaryotes that utilize cyclic di-AMP, and that it generates a strong innate immune response in eukaryotes. In bacteria that produce the second messenger, most molecular targets of cyclic di-AMP are associated with cell volume control. Besides, other evidence links the second messenger to cell wall remodeling, DNA damage repair, sporulation, central metabolism, and the regulation of glycogen turnover. In this review, we take a biochemical, quantitative approach to address the main cellular processes that are directly regulated by cyclic di-AMP and show that these processes are very connected and require regulation of a similar set of proteins to which cyclic di-AMP binds. Altogether, we argue that cyclic di-AMP is a master regulator of cell volume and that other cellular processes can be connected with cyclic di-AMP through this core function. We further highlight important directions in which the cyclic di-AMP field has to develop to gain a full understanding of the cyclic di-AMP signaling network and why some processes are regulated, while others are not.
Topics: Bacteria; Second Messenger Systems; Signal Transduction; Bacterial Proteins; Gene Expression Regulation, Bacterial; Dinucleoside Phosphates; Cell Wall
PubMed: 38856222
DOI: 10.1128/mmbr.00181-23 -
Current Diabetes Reports Jun 2023Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and... (Review)
Review
PURPOSE OF REVIEW
Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and middle-income countries, which are predominant in Latin America. Therefore, the purpose of this article is to compare the clinical practice guideline (CPG) for the pharmacological management of T2DM in Latin America (LA) with international reference guidelines.
RECENT FINDINGS
Several LA countries have recently developed CPGs. However, the quality of these guidelines is unknown according to the AGREE II tool and taking as reference three CPGs of international impact: American Diabetes Association (ADA), European Diabetes Association (EASD), and Latin American Diabetes Association (ALAD). Ten CPGs were selected for analysis. The ADA scored > 80% on the AGREE II domains and was selected as the main comparator. Eighty percent of LA CPGs were developed before 2018. Only one was not recommended (all domains < 60%). The CPGs in LA have good quality but are outdated. They have significant gaps compared to the reference. There is a need for improvement, as proposing updates every three years to maintain the best available clinical evidence in all guidelines.
Topics: Humans; Diabetes Mellitus, Type 2; Latin America; Dinucleoside Phosphates
PubMed: 37126189
DOI: 10.1007/s11892-023-01504-4 -
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 -
ACS Chemical Biology Sep 2021Many pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid, are potent immunostimulatory molecules and promote the...
Many pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid, are potent immunostimulatory molecules and promote the expression of cyclooxygenase 2 (COX-2). While the production of COX-2, and ultimately prostaglandin E, could be protective, persistent induction of COX-2 leads to inflamed environments that can result in septic shock and death. Bacterial derived cyclic dinucleotides (CDNs), c-di-GMP and c-di-AMP, are also PAMPs and have been shown to produce inflamed environments via the production of pro-inflammatory cytokines such as type I interferons. The well-characterized CDN immunostimulatory mechanism involves binding to stimulator of interferon genes (STING), which ultimately results in the phosphorylation of IRF3 or release of NF-κB to promote expression of type I IFN or pro-inflammatory cytokines. In this study, we sought to investigate if CDNs promote COX-2 expression. Using RAW macrophages as a model system, we reveal that c-di-GMP, but not c-di-AMP or the host-derived 2',3'-cGAMP, promotes COX-2 expression. Using analogues of CDNs, we show that the presence of two guanines and two 3',5'-phosphodiester linkages are requirements for the promotion of COX-2 expression by cyclic dinucleotides. Both c-di-GMP and LPS inductions of COX-2 expression in RAW macrophages are STING-independent and are regulated by Tpl2-MEK-ERK-CREB signaling; inhibitors of Tpl2, MEK, and ERK could attenuate COX-2 expression promoted by c-di-GMP. This work adds to the growing body of evidence that cyclic dinucleotides regulate pathways other than the STING-TBK1-IRF3 axis. Additionally, the differential COX-2 induction by c-di-GMP but not c-di-AMP or cGAMP suggests that the type and level of inflammation could be dictated by the nucleotide signature of the invading pathogen.
Topics: Animals; Beclin-1; Cell Line; Cyclic GMP; Cyclooxygenase 2; Dinucleoside Phosphates; Gene Expression Regulation; Guanine; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Mice; NF-kappa B; Oligonucleotides; Phosphorylation; Prostaglandins; Signal Transduction
PubMed: 34478263
DOI: 10.1021/acschembio.1c00342 -
Science Signaling Sep 2022The second messenger c-di-AMP contributes to various homeostatic and stress responses in bacteria. In this issue of , Oberkampf . have identified it as a mediator of... (Review)
Review
The second messenger c-di-AMP contributes to various homeostatic and stress responses in bacteria. In this issue of , Oberkampf . have identified it as a mediator of osmotic stress and bile salt resistance in the opportunistic pathogen , with additional roles in cell wall homeostasis and biofilm formation.
Topics: Bacterial Proteins; Clostridioides; Clostridioides difficile; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial
PubMed: 36067335
DOI: 10.1126/scisignal.add3937 -
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 Microbiology Sep 2022Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the...
Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.
Topics: Bacillus subtilis; Dinucleoside Phosphates; Guanosine Triphosphate
PubMed: 35953658
DOI: 10.1038/s41564-022-01193-x