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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 -
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 -
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 -
Cell Reports Apr 2023Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage...
Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.
Topics: Bacteria; Bacteriophages; Dinucleoside Phosphates; Guanosine Pentaphosphate; Immunity; Nucleotides; Nucleotidyltransferases
PubMed: 36952342
DOI: 10.1016/j.celrep.2023.112305 -
Scientific Reports Feb 2023Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response...
Inhibition of nicotinamide dinucleotide salvage pathway counters acquired and intrinsic poly(ADP-ribose) polymerase inhibitor resistance in high-grade serous ovarian cancer.
Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response to chemotherapy. Although poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promise for ovarian cancer treatment, extended therapy usually leads to acquired PARPi resistance. Here we explored a novel therapeutic option to counter this phenomenon, combining PARPi and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). Cell-based models of acquired PARPi resistance were created through an in vitro selection procedure. Using resistant cells, xenograft tumors were grown in immunodeficient mice, while organoid models were generated from primary patient tumor samples. Intrinsically PARPi-resistant cell lines were also selected for analysis. Our results show that treatment with NAMPT inhibitors effectively sensitized all in vitro models to PARPi. Adding nicotinamide mononucleotide, the resulting NAMPT metabolite, abrogated the therapy-induced cell growth inhibition, demonstrating the specificity of the synergy. Treatment with olaparib (PARPi) and daporinad (NAMPT inhibitor) depleted intracellular NAD+ , induced double-strand DNA breaks, and promoted apoptosis as monitored by caspase-3 cleavage. The two drugs were also synergistic in mouse xenograft models and clinically relevant patient-derived organoids. Therefore, in the context of PARPi resistance, NAMPT inhibition could offer a promising new option for ovarian cancer patients.
Topics: Humans; Animals; Mice; Female; Poly(ADP-ribose) Polymerase Inhibitors; Niacinamide; Antineoplastic Agents; Ovarian Neoplasms; Dinucleoside Phosphates
PubMed: 36849518
DOI: 10.1038/s41598-023-30081-5 -
Protein Science : a Publication of the... Mar 2023Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate...
Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.
Topics: Mycobacterium smegmatis; Bacterial Proteins; Dinucleoside Phosphates; Bacillus subtilis
PubMed: 36660887
DOI: 10.1002/pro.4568 -
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