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Biochemistry Aug 2023Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how...
Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.
Topics: Escherichia coli; Adenylate Kinase; Hydrolysis; Dinucleoside Phosphates; Catalysis; Catalytic Domain
PubMed: 37418448
DOI: 10.1021/acs.biochem.3c00189 -
Nature Communications Jun 2023Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In...
Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K/H symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K concentrations, further K accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K binding site and therefore traps KimA in an inward-occluded conformation.
Topics: Cyclic AMP; Protons; Bacterial Proteins; Second Messenger Systems; Membrane Transport Proteins; Potassium; Dinucleoside Phosphates
PubMed: 37344476
DOI: 10.1038/s41467-023-38944-1 -
Nucleic Acids Research Jul 2023The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World....
The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.
Topics: RNA; Oligonucleotides; RNA, Catalytic; Nucleotides; Dinucleoside Phosphates
PubMed: 37247941
DOI: 10.1093/nar/gkad439 -
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 -
Dalton Transactions (Cambridge, England... Feb 2023The first-in-class luminescent dinucleoside phosphate analogs with a [Re(μ-Cl)(CO)(μ-pyridazine)] "click" linker as a replacement for the natural phosphate group are...
Replacement of the phosphodiester backbone between canonical nucleosides with a dirhenium carbonyl "click" linker-a new class of luminescent organometallic dinucleoside phosphate mimics.
The first-in-class luminescent dinucleoside phosphate analogs with a [Re(μ-Cl)(CO)(μ-pyridazine)] "click" linker as a replacement for the natural phosphate group are reported together with the synthesis of luminescent adenosine and thymidine derivatives having the [Re(μ-Cl)(CO)(μ-pyridazine)] entity attached to positions 5' and 3', respectively. These compounds were synthesized by applying inverse-electron-demand Diels-Alder and copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reactions in three or four steps. The obtained compounds exhibited orange emission ( ≈ 600 nm, ≈ 0.10, and = 0.33-0.61 μs) and no toxicity (except for one nucleoside) to human HeLa cervical epithelioid and Ishikawa endometrial adenocarcinoma cancer cells . Furthermore, the compounds' ability to inhibit the growth of Gram-positive and Gram-negative bacterial strains was moderate and only observed at a high concentration of 100 μM. Confocal microscopy imaging revealed that the "dirhenium carbonyl" dinucleosides and nucleosides localized mainly in the membranous structures of HeLa cells and uniformly inside and bacterial cells. An interesting finding was that some of the tested compounds were also found in the nuclei of HeLa cells.
Topics: Humans; Nucleosides; HeLa Cells; Dinucleoside Phosphates; Phosphates; Escherichia coli; Staphylococcus aureus; Pyridazines; Click Chemistry
PubMed: 36655722
DOI: 10.1039/d2dt03995h