RESUMO
P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X7 is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X7 activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X7 bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na+ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.
Assuntos
Trifosfato de Adenosina , Microscopia Crioeletrônica , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Animais , Ratos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sítios de Ligação , Sódio/metabolismo , Humanos , Agonistas do Receptor Purinérgico P2X/farmacologia , Células HEK293 , Ligação Proteica , Modelos MolecularesRESUMO
Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1ß, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10-11 M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1ß and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats.
Assuntos
Citocinas , Indanos , Lipopolissacarídeos , Polifosfatos , Animais , Camundongos , Ratos , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fatores Imunológicos , Indanos/farmacologia , Interleucina-6 , Lipopolissacarídeos/farmacologia , Microglia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologiaRESUMO
The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Genoma , Diferenciação Sexual , Masculino , Humanos , Diferenciação Sexual/genética , Tionucleosídeos , CromossomosRESUMO
The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.
Assuntos
Trifosfato de Adenosina , Ensaios Enzimáticos , Miosina não Muscular Tipo IIA , Suínos , ortoaminobenzoatos , Animais , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Benzilaminas/farmacologia , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Ventrículos do Coração/metabolismo , Contração Miocárdica , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , ortoaminobenzoatos/metabolismo , Uracila/análogos & derivados , Uracila/farmacologiaRESUMO
Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers1-5, and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing protein 3 (NLRP3) is an inflammasome sensor of membrane damage highly important in regard to the induction of inflammation2,6,7. Here we report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with adenosine 5'-O-(3-thio)triphosphate, the centrosomal NIMA-related kinase 7 (NEK7) and the adaptor protein ASC, which recruits caspase-1. In these NLRP3-NEK7-ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by about 85° relative to the ADP-bound inactive conformation8-12. The fish-specific NACHT-associated domain conserved in NLRP3 but absent in most NLRPs13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disc. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all NLRP3 subunits combine to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disc interfaces. Together with previous structures of an inactive NLRP3 cage in which LRR-LRR interactions play an important role8-11, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disc.
Assuntos
Microscopia Crioeletrônica , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Caspase 1/metabolismo , Inflamassomos/química , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Quinases Relacionadas a NIMA , Domínios ProteicosAssuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Doenças Cardiovasculares/tratamento farmacológico , Trifosfato de Adenosina/análogos & derivados , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Hipolipemiantes/administração & dosagem , Doenças Cardiovasculares/prevenção & controle , Ensaios Clínicos Controlados Aleatórios como Assunto , Acidente Vascular Cerebral/epidemiologiaRESUMO
Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.
Assuntos
Trifosfato de Adenosina , Antivirais , SARS-CoV-2 , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Alanina/química , Antivirais/química , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus , Desoxirribonucleotídeos , Hidrogênio , Nucleotídeos , RNA Viral/genética , Ribonucleotídeos , SARS-CoV-2/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3'-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.
Assuntos
Cálcio , Gânglios Espinais , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Camundongos , Neuroglia , Neurônios/fisiologiaRESUMO
Purinergic receptors stimulation by adenosine triphosphate (ATP) contributes significantly to macrophage activation, and also macrophage cell death. Upon the macrophage activation, the protein load of the endoplasmic reticulum is increased which is resulted in the activation of unfolded protein response (UPR). In the current study, we aimed to evaluate the connection between prototypic P2X7 receptor agonist, extracellular 2'(3')-O-(4-Benzoylbenzoyl)-ATP (BzATP), and the UPR pathway in macrophages. The monocyte-derived macrophages from blood samples of 14 healthy volunteers were skewed toward M1 macrophages after incubation with LPS and IFN-γ. M1 macrophages were treated with 200 µM BzATP. The expression levels of UPR genes, including CHOP, HERP, GADD34, XBP1, and ATF6 in macrophages before and after treatment were measured using real-time polymerase chain reaction. The results demonstrated that the expression of CHOP, HERP, and ATF6 is significantly decreased and the expression level of GADD34 and XBP1 is significantly increased after M1 polarization. BzATP not only significantly increased the expression levels of CHOP, GADD34, ATF6, and HERP but also significantly decreases the XBP1 expression level in M1 macrophages. The present study showed that BzATP induces cellular stress in M1 macrophages by elevating the expression levels of UPR genes including CHOP, GADD34, ATF6, and reducing cell viability.
Assuntos
Trifosfato de Adenosina , Macrófagos , Agonistas do Receptor Purinérgico P2X , Resposta a Proteínas não Dobradas , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Humanos , Macrófagos/metabolismo , Agonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismoRESUMO
Calcium-dependent, neuronal adenylyl cyclase subtype 1 (AC1) is critical for cortical potentiation and chronic pain. NB001 is a first-in-class drug acting as a selective inhibitor against AC1. The present study delineated the pharmacokinetic (PK) properties of human-used NB001 (hNB001) formulated as immediate-release tablet. This first-in-human (FIH) study was designed as randomized, double-blind, placebo-controlled trial. hNB001 showed placebo-like safety and good tolerability in healthy volunteers. A linear dose-exposure relationship was demonstrated at doses between 20 mg and 400 mg. The relatively small systemic exposure of hNB001 in human showed low bioavailability of this compound through oral administration, which can be improved through future dosage research. Food intake had minimal impact on the absorption of hNB001 tablet. Animal experiments further confirmed that hNB001 had strong analgesic effect in animal models of neuropathic pain. In brain slice prepared from the anterior cingulate cortex (ACC), bath application of hNB001 blocked the induction of long-term potentiation (LTP). These results from both rodents and human strongly suggest that hNB001 can be safely used for the future treatment of different types of chronic pain in human patients.
Assuntos
Trifosfato de Adenosina , Inibidores de Adenilil Ciclases , Adenilil Ciclases , Dor Crônica , Neuralgia , Trifosfato de Adenosina/administração & dosagem , Trifosfato de Adenosina/efeitos adversos , Trifosfato de Adenosina/análogos & derivados , Inibidores de Adenilil Ciclases/administração & dosagem , Inibidores de Adenilil Ciclases/efeitos adversos , Adenilil Ciclases/metabolismo , Dor Crônica/tratamento farmacológico , Dor Crônica/enzimologia , Giro do Cíngulo/metabolismo , Humanos , Neuralgia/tratamento farmacológico , Neuralgia/enzimologiaRESUMO
The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.
Assuntos
Trifosfato de Adenosina/análogos & derivados , DNA Helicases/metabolismo , Complexos Multiproteicos/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA/química , DNA Helicases/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1-3 µg mL-1) or induce dye uptake alone at higher concentrations (10-20 µg mL-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Doenças Neuroinflamatórias , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Desenvolvimento de Medicamentos , Células HEK293 , Humanos , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Porinas/metabolismo , Agonistas Purinérgicos/farmacologia , Antagonistas do Receptor Purinérgico P2X/classificação , Antagonistas do Receptor Purinérgico P2X/farmacologia , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Células THP-1 , CatelicidinasRESUMO
The P2X7 receptor (P2X7R) is a calcium-permeable cation channel activated by high concentrations of extracellular ATP. It plays a role in vital physiological processes, particularly in innate immunity, and is dysregulated in pathological conditions such as inflammatory diseases, neurodegenerative diseases, mood disorders, and cancers. Structural modeling of the human P2X7R (hP2X7R) based on the recently available structures of the rat P2X7 receptor (rP2XR) in conjunction with molecular docking predicts the orientation of tyrosine at position 288 (Y288) in the extracellular domain to face ATP. In this short communication, we combined site-directed mutagenesis and whole-cell patch-clamp recording to investigate the role of this residue in the hP2X7R function. Mutation of this extracellular residue to amino acids with different properties massively impaired current responses to both ATP and BzATP, suggesting that Y288 is important for normal receptor function. Such a finding facilitates development of an in-depth understanding of the molecular basis of hP2X7R structure-function relationships.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Receptores Purinérgicos P2X7/química , Tirosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Humanos , Simulação de Acoplamento Molecular , Mutação , Técnicas de Patch-Clamp , Ligação Proteica , RatosRESUMO
ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced-but did not disappear altogether-in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Células Ciliadas Auditivas/metabolismo , Indóis/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Citocalasina D/farmacologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miosinas/metabolismo , Estereocílios/metabolismo , Estereocílios/ultraestrutura , Tiazolidinas/farmacologia , Vanadatos/farmacologiaRESUMO
SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , RNA Viral/biossíntese , SARS-CoV-2/enzimologia , Trifosfato de Adenosina/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Genoma Viral/genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Nucleotidiltransferases/metabolismo , Capuzes de RNA/genética , SARS-CoV-2/genética , Vaccinia virus/enzimologia , Vaccinia virus/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons and severe muscle atrophy without effective treatment. Most research on the disease has been focused on studying motor neurons and supporting cells of the central nervous system. Strikingly, the recent observations have suggested that morpho-functional alterations in skeletal muscle precede motor neuron degeneration, bolstering the interest in studying muscle tissue as a potential target for the delivery of therapies. We previously showed that the systemic administration of the P2XR7 agonist, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (BzATP), enhanced the metabolism and promoted the myogenesis of new fibres in the skeletal muscles of SOD1G93A mice. Here we further corroborated this evidence showing that intramuscular administration of BzATP improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of infiltrating macrophages. The preservation of the skeletal muscle retrogradely propagated along with the motor unit, suggesting that backward signalling from the muscle could impinge on motor neuron death. In addition to providing the basis for a suitable adjunct multisystem therapeutic approach in ALS, these data point out that the muscle should be at the centre of ALS research as a target tissue to address novel therapies in combination with those oriented to the CNS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Atividade Motora/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/administração & dosagem , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Axônios/patologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Denervação , Modelos Animais de Doenças , Progressão da Doença , Feminino , Membro Posterior/patologia , Humanos , Inflamação/patologia , Injeções Intramusculares , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Atrofia Muscular/patologia , Fenótipo , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologia , Células de Schwann/patologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologiaRESUMO
Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Morfolinas/química , Morfolinas/metabolismo , Mutação , Neoplasias/genética , Conformação Proteica , Pironas/química , Pironas/metabolismoRESUMO
Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) is one of the diffusible messengers for enhancing synaptic transmission in the hippocampus. Less information is available about the possible roles of BDNF in the anterior cingulate cortex (ACC). In the present study, we used 64-electrode array field recording system to investigate the effect of BDNF on ACC excitatory transmission. We found that BDNF enhanced synaptic responses in a dose-dependent manner in the ACC in C57/BL6 mice. The enhancement was long-lasting, and persisted for at least 3 h. In addition to the enhancement, BDNF also recruited inactive synaptic responses in the ACC. Bath application of the tropomyosin receptor kinase B (TrkB) receptor antagonist K252a blocked BDNF-induced enhancement. L-type voltage-gated calcium channels (L-VGCC), metabotropic glutamate receptors (mGluRs), but not NMDA receptors were required for BDNF-produced enhancement. Moreover, calcium-stimulated adenylyl cyclase subtype 1 (AC1) but not AC8 was essential for the enhancement. A selective AC1 inhibitor NB001 completely blocked the enhancement. Furthermore, BDNF-produced enhancement occluded theta burst stimulation (TBS) induced long-term potentiation (LTP), suggesting that they may share similar signaling mechanisms. Finally, the expression of BDNF-induced enhancement depends on postsynaptic incorporation of calcium-permeable AMPA receptors (CP-AMPARs) and protein kinase Mζ (PKMζ). Our results demonstrate that cortical BDNF may contribute to synaptic potentiation in the ACC.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Giro do Cíngulo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adenilil Ciclases/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Carbazóis/farmacologia , Relação Dose-Resposta a Droga , Eletrodos Implantados , Alcaloides Indólicos/farmacologia , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/fisiologia , Receptores de AMPA/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Sinapses/fisiologia , Ritmo Teta/efeitos dos fármacosRESUMO
Macrophages participate in the pathogenesis of ankylosing spondylitis (AS) by producing inflammatory cytokines. Extracellular adenosine triphosphate (eATP), released during cell stress, acts through purinergic receptors (P2XR and P2YR) and induces inflammatory responses. We investigated the effect of 2'(3')-O-(4-benzoyl benzoyl) ATP (BzATP) (a prototypic agonist of P2X7R) on the production of inflammatory cytokines in both monocyte-generated (M2-like) and M1 macrophages from patients and controls. Macrophages were differentiated from isolated periphery-monocytes (n = 14 in each group) by macrophage colony-stimulating factor (M-CSF). Using LPS and IFN-γ, macrophages were skewed toward M1 type and were treated with BzATP. Gene expression and protein release of IL-1ß, IL-23, and TNF-α were evaluated by real-time PCR and ELISA methods respectively before and after treatment. BzATP significantly increased the protein release of TNF-α and the expression of TNFA and IL1B in monocyte-generated macrophages. Besides, BzATP treatment significantly upregulated IL1B expression, reduced TNFA and IL23A expression, and TNF-α release in M1 macrophages from both groups. Monocyte-generated and M1 macrophages from AS patients released higher TNF-α and expressed more IL1B in response to the same concentration of BzATP treatment respectively. Based on our results, AS macrophages were more sensitive to BzATP treatment and responded more intensively. Besides, the diverse effects of BzATP on monocyte-derived and M1 macrophages in our study may represent the differed inflammatory properties of these two groups of macrophages in response to eATP in the body.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Espondilite Anquilosante/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Adulto , Feminino , Humanos , Interleucina-1beta/genética , Macrófagos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/efeitos dos fármacos , Adulto JovemRESUMO
A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.