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The Journal of Biological Chemistry Oct 2019IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide biosynthetic pathway. Because of its involvement in the...
IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.
Topics: Allosteric Regulation; Bacterial Infections; Binding Sites; Catalysis; Crystallography, X-Ray; Dinucleoside Phosphates; Eremothecium; Guanine Nucleotides; Humans; IMP Dehydrogenase; Models, Molecular; Neoplasms; Protein Conformation; Protein Domains; Scattering, Small Angle; X-Ray Diffraction
PubMed: 31416831
DOI: 10.1074/jbc.AC119.010055 -
Purinergic Signalling Apr 2024During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated...
During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (ApA), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that ApA negatively modulates the dendrite's growth and number by inducing transient intracellular calcium increases in the dendrites' growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of ApA on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by ApA. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by ApA, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that ApA exerts a negative influence on dendritic growth.
Topics: Adenosine Triphosphate; Receptors, Purinergic P2; Neurons; Dendrites; Hippocampus; Dinucleoside Phosphates
PubMed: 37246192
DOI: 10.1007/s11302-023-09944-z -
PLoS Genetics Jan 2021In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific...
In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.
Topics: Bacillus subtilis; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Glutamic Acid; Homeostasis; Ion Transport; Mutation; Potassium; Second Messenger Systems
PubMed: 33481774
DOI: 10.1371/journal.pgen.1009092 -
ELife Sep 2021Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network...
Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.
Topics: Bacterial Proteins; Binding Sites; Biofilms; Catalysis; Dinucleoside Phosphates; Enzyme Activation; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation; Pseudomonas aeruginosa; Signal Transduction; Structure-Activity Relationship
PubMed: 34498587
DOI: 10.7554/eLife.67289 -
Frontiers in Cellular and Infection... 2021Tuberculosis (TB), caused by (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary...
Tuberculosis (TB), caused by (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary for preventing the prevalence of TB. In this study, a subunit vaccine of ESAT-6 formulated with c-di-AMP (ESAT-6:c-di-AMP) promoted mucosal and systemic immune responses in spleen and lung. ESAT-6:c-di-AMP inhibited the differentiations of CD8 T cells as well as macrophages, but promoted the differentiations of ILCs in lung. The co-stimulation also enhanced inflammatory cytokines production in MH-S cells. It was first revealed that ESAT-6 and c-di-AMP regulated autophagy of macrophages in different stages, which together resulted in the inhibition of Mtb growth in macrophages during early infection. After Mtb infection, the level of ESAT-6-specific immune responses induced by ESAT-6:c-di-AMP dropped sharply. Finally, inoculation of ESAT-6:c-di-AMP led to significant reduction of bacterial burdens in lungs and spleens of immunized mice. Our results demonstrated that subunit vaccine ESAT-6:c-di-AMP could elicit innate and adaptive immune responses which provided protection against Mtb challenge, and c-di-AMP as a mucosal adjuvant could enhance immunogenicity of antigen, especially for innate immunity, which might be used for new mucosal vaccine against TB.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; CD8-Positive T-Lymphocytes; Dinucleoside Phosphates; Immunity; Mice; Mycobacterium tuberculosis; Tuberculosis; Tuberculosis Vaccines; Vaccines, Subunit
PubMed: 33829000
DOI: 10.3389/fcimb.2021.647220 -
NPJ Biofilms and Microbiomes Jul 2022Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis....
Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3',5')-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pde) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pde and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe-microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.
Topics: Adenosine Monophosphate; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Homeostasis; Phosphoric Diester Hydrolases; Porphyromonas gingivalis; Virulence
PubMed: 35794154
DOI: 10.1038/s41522-022-00316-w -
Cell Chemical Biology Nov 2019Diadenosine polyphosphates (ApAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApA...
Diadenosine polyphosphates (ApAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApA concentrations increase, it was postulated that ApAs are alarmones triggering stress-adaptive processes. The major synthesis pathway of ApAs is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like ApA formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to ApA formation for the first time.
Topics: Biocatalysis; Chromatography, High Pressure Liquid; Dinucleoside Phosphates; Humans; Mass Spectrometry; Mutagenesis; Recombinant Proteins; Small Ubiquitin-Related Modifier Proteins; Ubiquitin; Ubiquitin-Activating Enzymes
PubMed: 31492597
DOI: 10.1016/j.chembiol.2019.08.004 -
The Journal of Infectious Diseases Mar 2020Stimulator of interferon genes (STING) is a key cytosolic receptor for small nucleotides and plays a key role in anticancer and antiviral immunity. Cyclic dinucleotide...
BACKGROUND
Stimulator of interferon genes (STING) is a key cytosolic receptor for small nucleotides and plays a key role in anticancer and antiviral immunity. Cyclic dinucleotide STING agonists may comprise a novel class of vaccine adjuvants capable of inducing cellular immune responses and protective efficacy against intracellular pathogens.
METHODS
We generated a recombinant Bacillus Calmette-Guérin ([BCG] BCG-disA-OE) that overexpresses the endogenous mycobacterial diadenylate cyclase gene and releases high levels of the STING agonist bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP). We used a 24-week guinea pig vaccination-Mycobacterium tuberculosis (M.tb.) challenge model to test the protective efficacy of BCG-disA-OE versus wild-type BCG and measured lung weights, pathology scores, and M.tb. organ colony-forming unit (CFU) counts.
RESULTS
BCG-disA-OE elicited significantly stronger tumor necrosis factor-α, interleukin (IL)-6, IL-1β, interferon (IFN) regulatory factor 3, and IFN-β levels than BCG-wild type (WT) in vitro in murine macrophages. In vivo in guinea pigs, we found that BCG-disA-OE reduced lung weights, pathology scores, and M.tb. CFU counts in lungs by 28% (P < .05), 34%, and 2.0 log10 CFU units (P < .05) compared with BCG-WT, respectively.
CONCLUSIONS
We report a strategy of delivering a STING agonist from within live BCG. Overproduction of the STING agonist c-di-AMP significantly enhanced the protective efficacy of BCG against pulmonary and extrapulmonary tuberculosis. Our findings support the development of BCG-vectored STING agonists as a tuberculosis vaccine strategy.
Topics: Animals; BCG Vaccine; Cells, Cultured; Cytokines; Dinucleoside Phosphates; Female; Guinea Pigs; Lung; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Tuberculosis, Pulmonary
PubMed: 30901058
DOI: 10.1093/infdis/jiz116 -
Cell Reports Jun 2021Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of...
Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of mutations and transcription factor binding sites, we show that C/EBP binding sites are strongly enriched with [C > T]G mutations in cancer and adult stem cells, which is of special interest because C/EBPs regulate cell fate and differentiation. In vitro protein-DNA binding assay and structural modeling of the CEBPB-DNA complex show that the G·T mismatch in the core CG dinucleotide strongly enhances affinity of the binding site. We conclude that enhanced binding of C/EBPs shields CpG·TpG mismatches from DNA repair, leading to selective accumulation of [C > T]G mutations and consequent deterioration of the binding sites. This mechanism of targeted mutagenesis highlights the effect of a mutational process on certain regulatory sites and reveals the molecular basis of putative regulatory alterations in stem cells.
Topics: Adult Stem Cells; CCAAT-Enhancer-Binding Protein-alpha; Dinucleoside Phosphates; Humans; Mutation; Neoplasms
PubMed: 34107262
DOI: 10.1016/j.celrep.2021.109221 -
Viruses Apr 2020Distinct patterns of dinucleotide representation, such as CpG and UpA suppression, are characteristic of certain viral genomes. Recent research has uncovered vertebrate...
Distinct patterns of dinucleotide representation, such as CpG and UpA suppression, are characteristic of certain viral genomes. Recent research has uncovered vertebrate immune mechanisms that select against specific dinucleotides in targeted viruses. This evidence highlights the importance of systematically examining the dinucleotide composition of viral genomes. We have developed a novel metric, called synonymous dinucleotide usage (SDU), for quantifying dinucleotide representation in coding sequences. Our method compares the abundance of a given dinucleotide to the null hypothesis of equal synonymous codon usage in the sequence. We present a Python3 package, , for calculating SDU and other relevant metrics. We have applied this method on two sets of invertebrate- and vertebrate-specific flaviviruses and rhabdoviruses. The SDU shows that the vertebrate viruses exhibit consistently greater under-representation of CpG dinucleotides in all three codon positions in both datasets. In comparison to existing metrics for dinucleotide quantification, the SDU allows for a statistical interpretation of its values by comparing it to a null expectation based on the codon table. Here we apply the method to viruses, but coding sequences of other living organisms can be analysed in the same way.
Topics: Algorithms; Animals; Codon; Codon Usage; Dinucleoside Phosphates; Genome, Viral; Models, Theoretical; Species Specificity; Viruses
PubMed: 32325924
DOI: 10.3390/v12040462