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Scientific Reports Feb 2022The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA...
The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.
Topics: Animals; Base Sequence; Binding Sites; COVID-19; Dinucleoside Phosphates; Evolution, Molecular; Genome, Viral; Host-Pathogen Interactions; Humans; Nucleotide Motifs; Protein Binding; RNA, Viral; RNA-Binding Proteins; SARS-CoV-2; Virus Replication
PubMed: 35165300
DOI: 10.1038/s41598-022-06046-5 -
MBio Feb 2021In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of...
In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both and . This interaction stimulates the activity of the enzyme, as demonstrated by enzyme assays and metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.
Topics: Cyclic AMP; Bacillus subtilis; Pyruvate Carboxylase; Bacterial Proteins; Second Messenger Systems; Dinucleoside Phosphates; Glutamic Acid; Potassium
PubMed: 35130724
DOI: 10.1128/mbio.03602-21 -
Nature Immunology Feb 2022The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides...
The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c mice have increased cell cycle progression, proliferation, survival, Ca influx and cytokine production-a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING-p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity.
Topics: Animals; Anions; Calcium; Dinucleoside Phosphates; Female; Ion Channels; Membrane Proteins; Mice; Mice, Inbred C57BL; Nucleotides, Cyclic; Signal Transduction; T-Lymphocytes; Tumor Suppressor Protein p53
PubMed: 35105987
DOI: 10.1038/s41590-021-01105-x -
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 -
International Journal of Biological... 2022Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide...
Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both and , overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8 and CD4 T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5 CD8 T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.
Topics: Adjuvants, Immunologic; Animals; Dinucleoside Phosphates; Disease Models, Animal; Hepatitis B Vaccines; Hepatitis B, Chronic; Male; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides
PubMed: 34975324
DOI: 10.7150/ijbs.62424 -
International Journal of Molecular... Nov 2021(1) Background: is a brine shrimp containing high concentrations of dinucleotides, molecules with properties for dry eye treatment. For this reason, the purpose of the...
(1) Background: is a brine shrimp containing high concentrations of dinucleotides, molecules with properties for dry eye treatment. For this reason, the purpose of the study was to evaluate the effect of the artificial tears based on an extract of in a rabbit dry eye model. (2) Methods: A prospective and randomized study was carried out. Twenty rabbits were divided into 4 groups ( = 5, each group): healthy rabbits, dry eye rabbits, dry eye rabbits treated with hypromellose (HPMC), and dry eye rabbits treated with . Dry eye was induced by the topical instillation of 0.2% benzalkonium chloride. The measurements were performed before and after the treatment for 5 consecutive days. (3) Results: The topical instillation of artificial tears containing showed beneficial effects on tear secretion, tear break-up time, corneal staining, the density of Goblet cells, heigh of mucin cloud secreted by these cells, and mRNA levels of IL-1β and MMP9 in conjunctival cells. Compared with the HPMC, there was a statistically significant improvement ( < 0.05) with the in all the variables under study, except for the conjunctival hyperemia, density of Goblet cells, and mRNA levels of IL-6. (4) Conclusions: The potential of artificial tears based on as a secretagogue agent for dry eye treatment was confirmed, opening the door for future clinical trials and studies to extrapolate the findings for dry eye patients.
Topics: Animals; Artemia; Dinucleoside Phosphates; Disease Models, Animal; Dry Eye Syndromes; Hypromellose Derivatives; Lubricant Eye Drops; Male; Plant Extracts; Rabbits; Tears
PubMed: 34769429
DOI: 10.3390/ijms222111999 -
International Journal of Molecular... Oct 2021DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA...
DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.
Topics: Adenosine Triphosphatases; Bacillus subtilis; Bacterial Proteins; Chromosome Breakage; DNA Helicases; DNA Replication; DNA, Bacterial; DNA, Cruciform; DNA-Binding Proteins; Dinucleoside Phosphates; Escherichia coli; Holliday Junction Resolvases; Magnesium; Phosphorus-Oxygen Lyases
PubMed: 34768753
DOI: 10.3390/ijms222111323 -
International Journal of Molecular... Oct 2021Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several...
Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with K below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, mGppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.
Topics: Binding Sites; Circular Dichroism; Dinucleoside Phosphates; Humans; Hydrolysis; Kinetics; Molecular Docking Simulation; Protein Stability; Pyrophosphatases; Substrate Specificity; Thermodynamics
PubMed: 34681586
DOI: 10.3390/ijms222010929 -
Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment.Cell Oct 2021The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we...
The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we show that microbiota signals program mononuclear phagocytes in the TME toward immunostimulatory monocytes and dendritic cells (DCs). Single-cell RNA sequencing revealed that absence of microbiota skews the TME toward pro-tumorigenic macrophages. Mechanistically, we show that microbiota-derived stimulator of interferon genes (STING) agonists induce type I interferon (IFN-I) production by intratumoral monocytes to regulate macrophage polarization and natural killer (NK) cell-DC crosstalk. Microbiota modulation with a high-fiber diet triggered the intratumoral IFN-I-NK cell-DC axis and improved the efficacy of immune checkpoint blockade (ICB). We validated our findings in individuals with melanoma treated with ICB and showed that the predicted intratumoral IFN-I and immune compositional differences between responder and non-responder individuals can be transferred by fecal microbiota transplantation. Our study uncovers a mechanistic link between the microbiota and the innate TME that can be harnessed to improve cancer therapies.
Topics: Akkermansia; Animals; Dendritic Cells; Dietary Fiber; Dinucleoside Phosphates; Humans; Immune Checkpoint Inhibitors; Immunomodulation; Interferon Type I; Killer Cells, Natural; Macrophages; Melanoma; Membrane Proteins; Mice, Inbred BALB C; Mice, Inbred C57BL; Microbiota; Monocytes; Phagocytes; Transcription, Genetic; Tumor Microenvironment; Mice
PubMed: 34624222
DOI: 10.1016/j.cell.2021.09.019 -
Nature Communications Oct 2021The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase...
The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of ApA and ApA and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApAs across the domains of life, but also identify those that are different for ApA or ApA. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Topics: Adenosine Triphosphate; Dinucleoside Phosphates; Endoribonucleases; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; L-Lactate Dehydrogenase; Phosphoglycerate Kinase; Photoaffinity Labels; Protein Binding; Proteomics; Ubiquitin-Activating Enzymes
PubMed: 34608152
DOI: 10.1038/s41467-021-26075-4