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1.
Nat Commun ; 12(1): 5545, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545070

RESUMO

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nucleotídeos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Homologia de Sequência de Aminoácidos , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , DNA de Cadeia Simples/metabolismo , Fluorescência , Interferometria , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Especificidade da Espécie
2.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502352

RESUMO

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.


Assuntos
Membrana Eritrocítica/efeitos dos fármacos , Bifenil Polibromatos/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Membrana Eritrocítica/metabolismo , Eritrócitos/efeitos dos fármacos , Retardadores de Chama/toxicidade , Voluntários Saudáveis , Humanos , Fluidez de Membrana/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Bifenil Polibromatos/química , Bifenil Polibromatos/toxicidade , Conformação Proteica/efeitos dos fármacos , Proteínas/farmacologia
3.
Invest Ophthalmol Vis Sci ; 62(12): 19, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34546325

RESUMO

Purpose: The functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally. Methods: Rat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1-M5) and examined functionally regarding intracellular [Ca2+] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied. Results: Expression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10-11-10-3 M) did not cause a significant increase in intracellular [Ca2+]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10-8-10-3 M) caused a concentration-dependent increase in intracellular [Ca2+] that could be blocked by the P2 antagonists PPADS and suramin. Conclusions: Primary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca2+] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.


Assuntos
Células Epiteliais/metabolismo , Aparelho Lacrimal/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Purinérgicos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Imuno-Histoquímica , Aparelho Lacrimal/citologia , Aparelho Lacrimal/efeitos dos fármacos , Masculino , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Ratos , Ratos Sprague-Dawley
4.
Nat Chem Biol ; 17(11): 1148-1156, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34556859

RESUMO

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.


Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Endorribonucleases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Desdobramento de Proteína/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo
5.
Sci Rep ; 11(1): 17842, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34497300

RESUMO

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 Jovem
6.
Cells ; 10(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34359830

RESUMO

Lipocalin-2 (LCN2), a small secretory glycoprotein, is upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 inhibits bacterial growth by iron sequestration and regulates the innate immune system. Inflammasome activates the inflammatory caspases leading to pyroptosis and cytokine maturation. This study examined the effects of inflammasome activation on LCN2 secretion in response to TLR signaling. The triggers of NLRP3 inflammasome activation attenuated LCN2 secretion while it induced interleukin-1ß in mouse macrophages. In mice, NLRP3 inflammasome activation inhibited TLR-mediated LCN2 secretion. The inhibition of NLRP3 triggers on LCN2 secretion was caused by the inhibited transcription and translation of LCN2. At the same time, no changes in the other cytokines and IκBζ, a well-known transcriptional factor of Lcn2 transcription, were observed. Overall, NLRP3 triggers are a regulator of LCN2 expression suggesting a new linkage of inflammasome activation and LCN2 secretion in the innate immunity.


Assuntos
Inflamassomos/imunologia , Interleucina-1beta/imunologia , Lipocalina-2/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Trifosfato de Adenosina/farmacologia , Animais , Feminino , Fêmur/citologia , Fêmur/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Interleucina-1beta/genética , Lipocalina-2/genética , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nigericina/farmacologia , Cultura Primária de Células , Células RAW 264.7 , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Tíbia/citologia , Tíbia/imunologia , Transcrição Genética
7.
J Evid Based Med ; 14(3): 232-256, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34388310

RESUMO

The ATP-binding cassette (ABC) transporter superfamily is one of the largest membrane protein families existing in wide spectrum of organisms from prokaryotes to human. ABC transporters are also known as efflux pumps because they mediate the cross-membrane transportation of various endo- and xenobiotic molecules energized by ATP hydrolysis. Therefore, ABC transporters have been considered closely to multidrug resistance (MDR) in cancer, where the efflux of structurally distinct chemotherapeutic drugs causes reduced itherapeutic efficacy. Besides, ABC transporters also play other critical biological roles in cancer such as signal transduction. During the past decades, extensive efforts have been made in understanding the structure-function relationship, transportation profile of ABC transporters, as well as the possibility to overcome MDR via targeting these transporters. In this review, we discuss the most recent knowledge regarding ABC transporters and cancer drug resistance in order to provide insights for the development of more effective therapies.


Assuntos
Antineoplásicos , Neoplasias , Transportadores de Cassetes de Ligação de ATP/farmacologia , Transportadores de Cassetes de Ligação de ATP/uso terapêutico , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/uso terapêutico , Antineoplásicos/uso terapêutico , Resistência a Múltiplos Medicamentos , Humanos , Neoplasias/tratamento farmacológico
8.
Angew Chem Int Ed Engl ; 60(37): 20196-20199, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34292655

RESUMO

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP-site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP-competitive inhibitors and myristoyl-site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP-competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP-competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation-selective ATP-site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP-competitive inhibitors in a series of biochemical and cellular assays.


Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/química , Domínio Catalítico/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/química
9.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205953

RESUMO

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.


Assuntos
Conexinas/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores Purinérgicos P2X7/genética , Gânglio Trigeminal/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Cultura Primária de Células , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Gânglio Trigeminal/efeitos dos fármacos
10.
Biochem Biophys Res Commun ; 569: 112-117, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34243066

RESUMO

P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.


Assuntos
Trifosfato de Adenosina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mutação de Sentido Incorreto , Oócitos/fisiologia , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Sítios de Ligação/genética , Feminino , Ativação do Canal Iônico/genética , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Fatores de Tempo , Xenopus laevis
11.
Biochem Pharmacol ; 192: 114689, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34274353

RESUMO

P2Y receptors (P2YRs) are a δ group of rhodopsin-like G protein-coupled receptors (GPCRs) with many essential functions in physiology and pathology, such as platelet aggregation, immune responses, neuroprotective effects, inflammation, and cellular proliferation. Thus, they are among the most researched therapeutic targets used for the clinical treatment of diseases (e.g., the antithrombotic drug clopidogrel and the dry eye treatment drug diquafosol). GPCRs transmit signals as dimers to increase the diversity of signalling pathways and pharmacological activities. Many studies have frequently confirmed dimerization between P2YRs and other GPCRs due to their functions in cardiovascular and cerebrovascular processes in vivo and in vitro. Recently, some P2YR dimers that dynamically balance physiological functions in the body were shown to be involved in effective signal transduction and exert pathological responses. In this review, we summarize the types, pharmacological changes, and active regulators of P2YR-related dimerization, and delineate new functions and pharmacological activities of P2YR-related dimers, which may be a novel direction to improve the effectiveness of medications.


Assuntos
Agonistas do Receptor Purinérgico P2Y/metabolismo , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Receptores Purinérgicos P2Y/química , Receptores Purinérgicos P2Y/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Humanos , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo
12.
Microb Cell Fact ; 20(1): 144, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301268

RESUMO

BACKGROUND: Adenosine 5'-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. RESULTS: The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10-300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. CONCLUSIONS: Our results indicate that eATP release is an early event during the intimate bacterial-fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal-bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites.


Assuntos
Trifosfato de Adenosina/metabolismo , Ascomicetos/metabolismo , Perileno/análogos & derivados , Fenol/metabolismo , Pseudomonas/metabolismo , Quinonas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Ascomicetos/efeitos dos fármacos , Citosol/metabolismo , Perileno/análise , Perileno/metabolismo , Fenol/análise , Pseudomonas/efeitos dos fármacos , Quinonas/análise , Espécies Reativas de Oxigênio/metabolismo
13.
mBio ; 12(3): e0142321, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34154407

RESUMO

The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Guanosina Tetrafosfato/farmacologia , SARS-CoV-2/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , COVID-19/tratamento farmacológico , Domínio Catalítico/fisiologia , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Humanos , Ribossomos/metabolismo
14.
Elife ; 102021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34009126

RESUMO

Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.


Assuntos
Trifosfato de Adenosina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X2/efeitos dos fármacos , Animais , Fluorometria , Cinética , Potenciais da Membrana , Microscopia de Fluorescência , Mutação , Domínios Proteicos , Ratos , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
15.
Cells ; 10(4)2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33920157

RESUMO

Hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of hair follicles from mice and humans and have been shown to differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and beating cardiac muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal-cord regeneration in mouse models, differentiating to Schwann cells and neurons in this process. HAP stem cells can be banked by cryopreservation and preserve their ability to differentiate. In the present study, we demonstrated that mouse HAP stem cells cultured in neural-induction medium can extensively differentiate to dopaminergic neurons, which express tyrosine hydroxylase and secrete dopamine. These results indicate that the dopaminergic neurons differentiated from HAP stem cells may be useful in the future to improve the symptoms of Parkinson's disease in the clinic.


Assuntos
Diferenciação Celular , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Folículo Piloso/citologia , Células-Tronco Pluripotentes/citologia , Tirosina 3-Mono-Oxigenase/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Proliferação de Células , Neurônios Dopaminérgicos/metabolismo , Camundongos Endogâmicos C57BL
16.
Eur J Pharmacol ; 904: 174115, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33901459

RESUMO

In this report we examined the effects of lidocaine on Ca2+ homeostasis of neuronal cells using microfluorimetric measurement of cytosolic Ca2+ with fura 2 as probe. In mouse neuroblastoma N2A cells, 10 mM lidocaine caused Ca2+ release from the cyclopiazonic acid (CPA)-dischargeable pool and abolished ATP-triggered Ca2+ release. Lidocaine-triggered Ca2+ release was not affected by xestospongin C (XeC), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor. N2A cells did not have functional ryanodine receptors (RYR) (absence of caffeine response) and we used differentiated NG108-15 cells (presence of caffeine response) for further experiments. Caffeine-triggered Ca2+ release was unaffected by a brief lidocaine exposure, but was eliminated after a prolonged treatment of lidocaine, suggesting lidocaine abolished caffeine action possibly not by interfering caffeine binding but via Ca2+ store depletion. Lidocaine-elicited Ca2+ release was unaffected by XeC or a high concentration of ryanodine, suggesting Ca2+ release was not via IP3R or RYR. Lidocaine did not affect nigericin-dischargeable lysosomal Ca2+ stores. Lastly, we observed that lidocaine suppressed CPA-induced store-operated Ca2+ influx in both N2A cells and differentiated NG108-15 cells. Our results suggest two novel actions of lidocaine in neuronal cells, namely, depletion of Ca2+ store (via an IP3R- and RYR-independent manner) and suppression of store-operated Ca2+ influx.


Assuntos
Anestésicos Locais/farmacologia , Cálcio/metabolismo , Lidocaína/farmacologia , Neurônios/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Cafeína/farmacologia , Linhagem Celular Tumoral , Citosol/metabolismo , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Compostos Macrocíclicos/farmacologia , Camundongos , Técnicas Analíticas Microfluídicas , Neurônios/efeitos dos fármacos , Oxazóis/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
17.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802689

RESUMO

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca2+ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.


Assuntos
Amidas/farmacologia , Etanolaminas/farmacologia , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Ácidos Palmíticos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Ratos , Receptor CB2 de Canabinoide/metabolismo , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
J Med Chem ; 64(8): 4891-4902, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33822617

RESUMO

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.


Assuntos
Sistema Nervoso Central/metabolismo , Antagonistas do Receptor Purinérgico P2X/síntese química , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Sistema Nervoso Central/efeitos dos fármacos , Cães , Feminino , Meia-Vida , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microssomos Hepáticos/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Antagonistas do Receptor Purinérgico P2X/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/química
19.
Cell Biochem Funct ; 39(5): 688-698, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33821520

RESUMO

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells.


Assuntos
Trifosfato de Adenosina/farmacologia , Angiotensina II/farmacologia , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Potássio/farmacologia , Prosencéfalo/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Masculino , Canais de Potássio/genética , Canais de Potássio/metabolismo , Prosencéfalo/efeitos dos fármacos , Ratos , Ratos Wistar
20.
Cell Biochem Biophys ; 79(2): 221-229, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33733369

RESUMO

Defects in inosine monophosphate dehydrogenase-1 (IMPDH1) lead to insufficient biosyntheses of purine nucleotides. In eyes, these defects are believed to cause retinitis pigmentosa (RP). Major retinal isoforms of IMPDH1 are structurally distinct from those in other tissues, by bearing terminal extensions. Using recombinant mouse IMPDH1 (mH1), we evaluated the kinetics and oligomerization states of the retinal isoforms. Moreover, we adopted molecular simulation tools to study the possible effect of terminal tails on the function of major enzyme isoforms with the aim to find structural evidence in favor of contradictory observations on retinal IMPDH1 function. Our findings indicated higher catalytic activity for the major mouse retinal isoform (mH1603) along with lower fibrillation capacity under the influence of ATP. However, higher mass oligomerization products were formed by the mH1 (603) isoform in the presence of the enzyme inhibitors such as GTP and/or MPA. Collectively, our findings demonstrate that the structural differences between the retinal isoforms have led to functional variations possibly to justify the retinal cells' requirements.


Assuntos
IMP Desidrogenase/metabolismo , Retina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Humanos , Ligação de Hidrogênio , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/química , IMP Desidrogenase/genética , Cinética , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
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