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1.
PLoS Pathog ; 20(8): e1012486, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39159286

RESUMO

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes a wide range of infections that are difficult to treat, largely because of the spread of antibiotic-resistant isolates. Antivirulence therapy, í.e. the use of drugs that inhibit the expression or activity of virulence factors, is currently considered an attractive strategy to reduce P. aeruginosa pathogenicity and complement antibiotic treatments. Because of the multifactorial nature of P. aeruginosa virulence and the broad arsenal of virulence factors this bacterium can produce, the regulatory networks that control the expression of multiple virulence traits have been extensively explored as potential targets for antivirulence drug development. The intracellular signaling molecule diadenosine tetraphosphate (Ap4A) has been reported to control stress resistance and virulence-related traits in some bacteria, but its role has not been investigated in P. aeruginosa so far. To fill this gap, we generated a mutant of the reference strain P. aeruginosa PAO1 that lacks the Ap4A-hydrolysing enzyme ApaH and, consequently, accumulates high intracellular levels of Ap4A. Phenotypic and transcriptomic analyses revealed that the lack of ApaH causes a drastic reduction in the expression of several virulence factors, including extracellular proteases, elastases, siderophores, and quorum sensing signal molecules. Accordingly, infection assays in plant and animal models demonstrated that ApaH-deficient cells are significantly impaired in infectivity and persistence in different hosts, including mice. Finally, deletion of apaH in P. aeruginosa clinical isolates demonstrated that the positive effect of ApaH on the production of virulence-related traits and on infectivity is conserved in P. aeruginosa. This study provides the first evidence that the Ap4A-hydrolysing enzyme ApaH is important for P. aeruginosa virulence, highlighting this protein as a novel potential target for antivirulence therapies against P. aeruginosa.


Assuntos
Fosfatos de Dinucleosídeos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Fatores de Virulência , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/genética , Animais , Camundongos , Virulência , Infecções por Pseudomonas/microbiologia , Fosfatos de Dinucleosídeos/metabolismo , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Hidrolases Anidrido Ácido/metabolismo , Hidrolases Anidrido Ácido/genética , Regulação Bacteriana da Expressão Gênica
2.
Acta Crystallogr F Struct Biol Commun ; 80(Pt 9): 200-209, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39177700

RESUMO

Crystallographic fragment screening has become a pivotal technique in structure-based drug design, particularly for bacterial targets with a crucial role in infectious disease mechanisms. The enzyme CdaA, which synthesizes an essential second messenger cyclic di-AMP (c-di-AMP) in many pathogenic bacteria, has emerged as a promising candidate for the development of novel antibiotics. To identify crystals suitable for fragment screening, CdaA enzymes from Streptococcus pneumoniae, Bacillus subtilis and Enterococcus faecium were purified and crystallized. Crystals of B. subtilis CdaA, which diffracted to the highest resolution of 1.1 Å, were used to perform the screening of 96 fragments, yielding data sets with resolutions spanning from 1.08 to 1.87 Å. A total of 24 structural hits across eight different sites were identified. Four fragments bind to regions that are highly conserved among pathogenic bacteria, specifically the active site (three fragments) and the dimerization interface (one fragment). The coordinates of the three active-site fragments were used to perform an in silico drug-repurposing screen using the OpenEye suite and the DrugBank database. This screen identified tenofovir, an approved drug, that is predicted to interact with the ATP-binding region of CdaA. Its inhibitory potential against pathogenic E. faecium CdaA has been confirmed by ITC measurements. These findings not only demonstrate the feasibility of this approach for identifying lead compounds for the design of novel antibacterial agents, but also pave the way for further fragment-based lead-optimization efforts targeting CdaA.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Bacillus subtilis/enzimologia , Cristalografia por Raios X/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Modelos Moleculares , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/química , Sequência de Aminoácidos , Ligação Proteica , Cristalização
3.
Curr Opin Microbiol ; 81: 102536, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39216180

RESUMO

Bacteria thrive in diverse environments and must withstand various stresses. A key stress response mechanism is the reprogramming of macromolecular biosynthesis and metabolic processes through alarmones - signaling nucleotides that accumulate intracellularly in response to metabolic stress. Diadenosine tetraphosphate (Ap4A), a putative alarmone, is produced in a noncanonical reaction by universally conserved aminoacyl-tRNA synthetases. Ap4A is ubiquitous across all domains of life and accumulates during heat and oxidative stress. Despite its early discovery in 1966, Ap4A's alarmone status remained inconclusive. Recent discoveries identified Ap4A as a precursor to RNA 5' caps in Escherichia coli. Additionally, Ap4A was found to directly bind to and allosterically inhibit the purine biosynthesis enzyme inosine 5'-monophosphate dehydrogenase, regulating guanosine triphosphate levels and enabling heat resistance in Bacillus subtilis. These findings, along with previous research, strongly suggest that Ap4A plays a crucial role as an alarmone, warranting further investigation to fully elucidate its functions.


Assuntos
Fosfatos de Dinucleosídeos , Proteostase , Estabilidade de RNA , Fosfatos de Dinucleosídeos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bactérias/metabolismo , Bactérias/genética , Estresse Fisiológico
4.
Vet Microbiol ; 297: 110194, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39084162

RESUMO

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host's innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3'3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3'3'-cGAMP and host-derived 2'3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2'3'-cGAMP. In addition, CpdB has 2'3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.


Assuntos
Imunidade Inata , Diester Fosfórico Hidrolases , Infecções por Yersinia pseudotuberculosis , Yersinia pseudotuberculosis , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/genética , Animais , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Infecções por Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos Cíclicos/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Fosfatos de Dinucleosídeos/metabolismo , Feminino , GMP Cíclico/análogos & derivados
5.
mBio ; 15(8): e0141124, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38980040

RESUMO

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.


Assuntos
Proteínas de Bactérias , Fosfatos de Dinucleosídeos , Regulação Bacteriana da Expressão Gênica , Homeostase , Acetilação , Fosfatos de Dinucleosídeos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Actinobacteria/metabolismo , Actinobacteria/genética , Organofosfatos/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética
6.
Microbiol Mol Biol Rev ; 88(2): e0018123, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38856222

RESUMO

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.


Assuntos
Bactérias , Bactérias/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfatos de Dinucleosídeos/metabolismo , Parede Celular/metabolismo
7.
J Bacteriol ; 206(7): e0019024, 2024 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-38832794

RESUMO

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.


Assuntos
Aminoácidos , Bacillus subtilis , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Homeostase , Pressão Osmótica , Potássio , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Potássio/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Aminoácidos/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Mutação
8.
Microbiol Spectr ; 12(8): e0378623, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-38899864

RESUMO

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.


Assuntos
Antígenos de Bactérias , Bacillus anthracis , Proteínas de Bactérias , Toxinas Bacterianas , Fosfatos de Dinucleosídeos , Regulação Bacteriana da Expressão Gênica , Potássio , Bacillus anthracis/metabolismo , Bacillus anthracis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Potássio/metabolismo , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Fosfatos de Dinucleosídeos/metabolismo , Virulência/genética , Regulação para Baixo , Antraz/microbiologia , Antraz/metabolismo , Riboswitch/genética , Óperon , Proteínas Quinases
9.
Front Cell Infect Microbiol ; 14: 1418651, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38933693

RESUMO

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.


Assuntos
Lipopolissacarídeos , Porphyromonas gingivalis , Transdução de Sinais , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Lipopolissacarídeos/metabolismo , Fatores de Virulência/metabolismo , Regulação Bacteriana da Expressão Gênica , Metabolismo Energético , Fosfatos de Dinucleosídeos/metabolismo , Ácidos Graxos/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética
10.
Structure ; 32(8): 1197-1207.e4, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38701795

RESUMO

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future.


Assuntos
Proteínas de Bactérias , Domínio Catalítico , Fosfatos de Dinucleosídeos , Modelos Moleculares , Streptococcus pneumoniae , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/metabolismo , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Especificidade por Substrato , Hidrolases/química , Hidrolases/metabolismo , Hidrolases/genética , Ligação Proteica , Sequência de Aminoácidos , Hidrólise , Sítios de Ligação
11.
Nat Commun ; 15(1): 3825, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714645

RESUMO

c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.


Assuntos
Acetilglucosamina , Proteínas de Bactérias , Fosfatos de Dinucleosídeos , Regulação Bacteriana da Expressão Gênica , Saccharopolyspora , Transdução de Sinais , Acetilglucosamina/metabolismo , Regulação Alostérica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fosfatos de Dinucleosídeos/metabolismo , Saccharopolyspora/metabolismo , Saccharopolyspora/genética
12.
Bioorg Chem ; 148: 107432, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38744169

RESUMO

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.


Assuntos
Adenilato Quinase , Fosfatos de Dinucleosídeos , Relação Quantitativa Estrutura-Atividade , Humanos , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/farmacologia , Fosfatos de Dinucleosídeos/metabolismo , Cinética , Estrutura Molecular , Adenilato Quinase/metabolismo , Adenilato Quinase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química
13.
Chemistry ; 30(41): e202401302, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38763895

RESUMO

Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through in situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.


Assuntos
Adenosina Difosfato Ribose , Trifosfato de Adenosina , Trifosfato de Adenosina/química , Adenosina Difosfato Ribose/química , Hidrólise , Difosfato de Adenosina/química , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Estrutura Molecular
14.
J Am Chem Soc ; 146(15): 10632-10639, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38579124

RESUMO

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.


Assuntos
RNA Catalítico , RNA , RNA/química , Metais , Fosfatos de Dinucleosídeos , Fosfatos , Catálise , Oxigênio , Íons , RNA Catalítico/química
15.
Proc Natl Acad Sci U S A ; 121(18): e2318666121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38652747

RESUMO

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Potássio/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfatos de Dinucleosídeos/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio/genética
16.
Acta Crystallogr D Struct Biol ; 80(Pt 5): 350-361, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38682668

RESUMO

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.


Assuntos
Listeria monocytogenes , Listeria monocytogenes/enzimologia , Cristalografia por Raios X/métodos , Sítios de Ligação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Fosfatos de Dinucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/química , Antibacterianos/farmacologia , Humanos , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica
17.
Mol Oral Microbiol ; 39(5): 354-367, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38436552

RESUMO

Pathobionts associated with periodontitis, such as Treponema denticola, must possess numerous sensory transduction systems to adapt to the highly dynamic subgingival environment. To date, the signaling pathways utilized by T. denticola to rapidly sense and respond to environmental stimuli are mainly unknown. Bis-(3'-5') cyclic diadenosine monophosphate (c-di-AMP) is a nucleotide secondary messenger that regulates osmolyte transport, central metabolism, biofilm development, and pathogenicity in many bacteria but is uncharacterized in T. denticola. Here, we studied c-di-AMP signaling in T. denticola to understand how it contributes to T. denticola physiology. We demonstrated that T. denticola produces c-di-AMP and identified enzymes that function in the synthesis (TDE1909) and hydrolysis (TDE0027) of c-di-AMP. To investigate how c-di-AMP may impact T. denticola cellular processes, a screening assay was performed to identify putative c-di-AMP receptor proteins. This approach identified TDE0087, annotated as a potassium uptake protein, as the first T. denticola c-di-AMP binding protein. As potassium homeostasis is critical for maintaining turgor pressure, we demonstrated that T. denticola c-di-AMP concentrations are impacted by osmolarity, suggesting that c-di-AMP negatively regulates potassium uptake in hypoosmotic solutions. Collectively, this study demonstrates T. denticola utilizes c-di-AMP signaling, identifies c-di-AMP metabolism proteins, identifies putative receptor proteins, and correlates c-di-AMP signaling to osmoregulation.


Assuntos
Proteínas de Bactérias , Biofilmes , Fosfatos de Dinucleosídeos , Transdução de Sinais , Treponema denticola , Treponema denticola/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Periodontite/microbiologia
18.
Biomater Sci ; 12(9): 2292-2301, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38498328

RESUMO

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.


Assuntos
Neoplasias Colorretais , Fosfatos de Dinucleosídeos , Nanopartículas , Tretinoína , Animais , Feminino , Humanos , Camundongos , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Imunoterapia/métodos , Nanopartículas em Multicamadas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Tretinoína/administração & dosagem , Tretinoína/farmacologia
19.
Org Biomol Chem ; 22(15): 3025-3034, 2024 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-38530278

RESUMO

Four dinucleotide analogs of thymidylyl(3'-5')thymidine (TpT) have been designed and synthesized with a view to increase the selectivity, with respect to CPD, of efficient UV-induced (6-4) photoproduct formation. The deoxyribose residues of these analogs have been modified to increase north and south conformer populations at 5'- and 3'-ends, respectively. Dinucleotides whose 5'-end north population exceeds ca. 60% and whose 3'-end population is almost completely south display a three-fold selective enhancement in (6-4) adduct production when exposed to UV radiation, compared to TpT. These experimental results undoubtedly provide robust foundations for studying the singular ground-state proreactive species involved in the (6-4) photoproduct formation mechanism.


Assuntos
Carboidratos , Açúcares , Fotoquímica , Carboidratos/química , Fosfatos de Dinucleosídeos/química , Raios Ultravioleta
20.
Nat Struct Mol Biol ; 31(5): 767-776, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38321146

RESUMO

The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS-STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) with second messengers. The key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.


Assuntos
Proteínas de Bactérias , Endonucleases , Pseudomonas syringae , Pseudomonas syringae/química , Pseudomonas syringae/enzimologia , Pseudomonas syringae/virologia , Proteínas de Bactérias/química , Endonucleases/química , Ligantes , Modelos Químicos , Ativação Enzimática , DNA/química , DNA/metabolismo , Nucleotídeos Cíclicos/química , Fosfatos de Dinucleosídeos/química , Apoproteínas/química , Bacteriófagos/fisiologia
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