RESUMO
The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed.
Assuntos
Cromossomos , Cromossomos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA/química , DNA/metabolismo , DNA/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/químicaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.
Assuntos
Sobrevivência Celular , Mitocôndrias , Neurônios , Fármacos Neuroprotetores , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Trifosfato de Adenosina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/farmacologia , Relação Dose-Resposta a Droga , Superóxidos/metabolismoRESUMO
Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.
Assuntos
Trifosfato de Adenosina , Carbenoxolona , Conexinas , Proteínas do Tecido Nervoso , Recuperação de Função Fisiológica , Corantes de Rosanilina , Traumatismos da Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Animais , Conexinas/metabolismo , Conexinas/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Carbenoxolona/farmacologia , Carbenoxolona/uso terapêutico , Corantes de Rosanilina/farmacologia , Corantes de Rosanilina/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Recuperação de Função Fisiológica/efeitos dos fármacos , Ratos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Ratos Sprague-Dawley , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/efeitos dos fármacos , Feminino , Infiltração de Neutrófilos/efeitos dos fármacosRESUMO
P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.
Assuntos
Trifosfato de Adenosina , Microscopia Crioeletrônica , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X1 , Humanos , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X1/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Ligantes , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ligação Proteica , Sítios de Ligação , Células HEK293 , Modelos Moleculares , BenzenossulfonatosRESUMO
Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration.
Assuntos
Axônios , Glicólise , Mitocôndrias , Regeneração Nervosa , Células Ganglionares da Retina , Animais , Axônios/metabolismo , Regeneração Nervosa/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Mitocôndrias/metabolismo , Mitocôndrias/genética , Camundongos , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Camundongos Endogâmicos C57BL , Trifosfato de Adenosina/metabolismo , Metabolismo Energético/genéticaRESUMO
A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.
Assuntos
Trifosfato de Adenosina , Fosfofrutoquinase-1 , Trifosfato de Adenosina/metabolismo , Fosfofrutoquinase-1/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Frutosefosfatos/metabolismo , Difosfato de Adenosina/metabolismo , GlicóliseRESUMO
The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.
Assuntos
Cinesinas , Microtúbulos , Tetrahymena thermophila , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo , Tetrahymena thermophila/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Trifosfato de Adenosina/metabolismo , Cílios/metabolismo , Tetrahymena/metabolismo , Tetrahymena/genéticaRESUMO
Our brain is not an immune-privileged island isolated from peripheries, but how non-neuronal brain cells interact with the peripheral system is not well understood. Wei et al. report that microglia in the hypothalamic paraventricular nucleus (PVN) with unique vasculature can detect ATP derived from hemodynamic disturbance. These microglia in the PVN regulate the response to hypertension via ATP-P2Y12-C/EBPß signaling.
Assuntos
Pressão Sanguínea , Encéfalo , Microglia , Núcleo Hipotalâmico Paraventricular , Microglia/imunologia , Microglia/fisiologia , Microglia/metabolismo , Animais , Humanos , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/imunologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Pressão Sanguínea/fisiologia , Encéfalo/imunologia , Trifosfato de Adenosina/metabolismo , Transdução de Sinais , Hipertensão/imunologia , Hipertensão/fisiopatologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismoRESUMO
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in almost all eukaryotic cells. In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the α1 (but not the α2) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Humanos , Ativação Enzimática/efeitos dos fármacos , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Animais , Fosforilação/efeitos dos fármacos , Trifosfato de Adenosina/metabolismoRESUMO
Bioenergy decline occurs with reperfusion following acute ischemic stroke. However, the molecular mechanisms that limit energy metabolism and their impact on post-stroke cognitive and emotional complications are still unclear. In the present study, we demonstrate that the p53 transcriptional response is responsible for neuronal adenosine triphosphate (ATP) deficiency and progressively neuropsychiatric disturbances, involving the downregulation of mitochondrial voltage-dependent anion channels (VDACs). Neuronal p53 transactivated the promoter of microRNA-183 (miR-183) cluster, thereby upregulating biogenesis of miR-183-5p (miR-183), miR-96-5p (miR-96), and miR-182-5p. Both miR-183 and miR-96 directly targeted and post-transcriptionally suppressed VDACs. Neuronal ablation of p53 protected against ATP deficiency and neurological deficits, whereas post-stroke rescue of miR-183/VDAC signaling reversed these benefits. Interestingly, cyclin-dependent kinase 9 (CDK9) was found to be enriched in cortical neurons and upregulated the p53-induced transcription of the miR-183 cluster in neurons after ischemia. Post-treatment with the CDK9 inhibitor oroxylin A promoted neuronal ATP production mainly through suppressing the miR-183 cluster/VDAC axis, further improved long-term sensorimotor abilities and spatial memory, and alleviated depressive-like behaviors in mice following stroke. Our findings reveal an intrinsic CDK9/p53/VDAC pathway that drives neuronal bioenergy decline and underlies post-stroke cognitive impairment and depression, thus highlighting the therapeutic potential of oroxylin A for better outcomes.
Assuntos
Metabolismo Energético , Camundongos Endogâmicos C57BL , MicroRNAs , Neurônios , Transdução de Sinais , Acidente Vascular Cerebral , Proteína Supressora de Tumor p53 , Animais , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/complicações , Trifosfato de Adenosina/metabolismoRESUMO
In the realm of this study, obtaining a comprehensive understanding of ischemic brain injury and its molecular foundations is of paramount importance. Our study delved into single-cell data analysis, with a specific focus on sub-celltypes and differentially expressed genes in the aftermath of ischemic injury. Notably, we observed a significant enrichment of the "ATP METABOLIC PROCESS" and "ATP HYDROLYSIS ACTIVITY" pathways, featuring pivotal genes such as Pbx3, Dguok, and Kif21b. A remarkable finding was the consistent upregulation of genes like Fabp7 and Bcl11a within the MCAO group, highlighting their crucial roles in regulating the pathway of mitochondrial ATP synthesis coupled proton transport. Furthermore, our network analysis unveiled pathways like "Neuron differentiation" and "T cell differentiation" as central in the regulatory processes of sub-celltypes. These findings provide valuable insights into the intricate molecular responses and regulatory mechanisms that govern brain injury. The shared differentially expressed genes among sub-celltypes emphasize their significance in orchestrating responses post-ischemic injury. Our research, viewed from the perspective of a medical researcher, contributes to the evolving understanding of the molecular landscape underlying ischemic brain injury, potentially paving the way for targeted therapeutic strategies and improved patient outcomes.
Assuntos
Trifosfato de Adenosina , Infarto da Artéria Cerebral Média , Cinesinas , Mitocôndrias , Células Precursoras de Oligodendrócitos , Transdução de Sinais , Animais , Transdução de Sinais/genética , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Cinesinas/genética , Cinesinas/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Humanos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Ratos , Proteínas Proto-OncogênicasRESUMO
The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.
Assuntos
Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Peptidilprolil Isomerase , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Peptidilprolil Isomerase/genética , Ligação Proteica , Domínios Proteicos , Dobramento de ProteínaRESUMO
AIMS: Anxiety often coexists with migraine, and both conditions share a commonality in oxidative/nitrosative stress and mitochondrial dysfunction contributing to their pathogenesis. ß-Sitosterol, a plant sterol, has shown promise in mitigating oxidative/nitrosative stress, enhancing mitochondrial function, and exerting neuroprotective effects. In this study, we investigated the impact of ß-sitosterol on migraine-associated anxiety and whether this effect was associated with alleviation of oxidative/nitrosative stress and improvement in mitochondrial function. METHODS: Nitroglycerin was used to induce migraine in adult male Wistar rats. ß-Sitosterol treatment consisted of daily intraperitoneal injections (10 mg/kg) for 10 days following migraine induction. Anxiety levels were evaluated using open-field test (OFT) and hole-board test (HBT). Frontal cortex samples were analyzed for malondialdehyde (MDA), glutathione (GSH), reactive oxygen/nitrogen species, nitric oxide (NO) (markers of oxidative/nitrosative stress), and ATP (indicator of mitochondrial function). RESULTS: Migraine induction led to impaired performance in both the OFT and the HBT. Concurrently, it elevated MDA, reactive oxygen/nitrogen species, and NO levels while diminishing GSH levels in the frontal cortex, signifying heightened oxidative/nitrosative stress. Moreover, ATP levels decreased, indicating mitochondrial dysfunction. Treatment with ß-sitosterol significantly restored performance in both behavioral assays and normalized the levels of MDA, GSH, reactive oxygen/nitrogen species, NO, and ATP. CONCLUSION: ß-Sitosterol exerted anxiolytic effects in migraine, which can be attributed to its ability to ameliorate oxidative/nitrosative stress and enhance mitochondrial function.
Assuntos
Ansiedade , Modelos Animais de Doenças , Transtornos de Enxaqueca , Mitocôndrias , Estresse Oxidativo , Ratos Wistar , Sitosteroides , Animais , Masculino , Sitosteroides/farmacologia , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ansiedade/tratamento farmacológico , Ratos , Malondialdeído/metabolismo , Nitroglicerina/farmacologia , Glutationa/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Teste de Campo Aberto/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismoRESUMO
New agrochemicals must demonstrate safety to numerous ecological systems, including aquatic systems, and aquatic vertebrate toxicity is typically evaluated by using the in vivo acute fish toxicity (AFT) test. Here, we investigated two alternative in vitro assays using a cell line isolated from rainbow trout (Onchorhynchus mykiss) gill tissue: (i) adenosine triphosphate (ATP) luminescence and (ii) cell painting. The former assay measures cytotoxicity, while the latter measures changes in cellular morphology in response to chemical exposure. We assessed how well end points in these two assays predicted acute lethality (i.e., LC50 values) in independent in vivo AFT tests. When compared to results from OECD TG 249 (in vitro), we found that the ATP assay was not as predictive (R2 = 0.53) as the cell painting assay. Similarly, when compared to results from OECD TG 203 (in vivo), the cell painting was much more predictive (R2 = 0.67). Our results show that such in vitro assays are useful for fast and efficient screening alternatives to in vivo fish testing that can aid in the agrochemical discovery phase, where thousands of potential new actives are tested each year.
Assuntos
Agroquímicos , Oncorhynchus mykiss , Animais , Agroquímicos/toxicidade , Proteção de Cultivos/métodos , Testes de Toxicidade Aguda , Linhagem Celular , Poluentes Químicos da Água/toxicidade , Trifosfato de Adenosina/metabolismoRESUMO
We isolated a stress-tolerance-related gene from a genome library of Synechococcus sp. NKBG15041c. The expression of the gene in E. coli confers resistance against various stresses. The gene encodes a MoxR AAA+ ATPase, which was designated SyMRP since it belongs to the MRP subfamily. The recombinant SyMRP showed weak ATPase activity and protected citrate synthase from thermal aggregation. Interestingly, the chaperone activity of SyMRP is ATP-dependent. SyMRP exists as a stable hexamer, and ATP-dependent conformation changes were not detected via analytical ultracentrifugation (AUC) or small-angle X-ray scattering (SAXS). Although the hexameric structure predicted by AlphaFold 3 was the canonical flat-ring structure, the structures observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) were not the canonical ring structure. In addition, the experimental SAXS profiles did not show a peak that should exist in the symmetric-ring structure. Therefore, SyMRP seems to form a hexameric structure different from the canonical hexameric structure of AAA+ ATPase.
Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Synechococcus , Synechococcus/enzimologia , Synechococcus/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Espalhamento a Baixo Ângulo , Difração de Raios X , Microscopia de Força Atômica , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [3H]-α,ß-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor.
Assuntos
Trifosfato de Adenosina , Microscopia Crioeletrônica , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X1 , Humanos , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X1/química , Receptores Purinérgicos P2X1/genética , Trifosfato de Adenosina/metabolismo , Ligantes , Antagonistas do Receptor Purinérgico P2X/farmacologia , Células HEK293 , Ligação Proteica , Sítios de Ligação , Modelos Moleculares , BenzenossulfonatosRESUMO
The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.
Assuntos
Trifosfato de Adenosina , Toxinas Bacterianas , Cálcio , Canais de Cloreto , Vesículas Extracelulares , Oócitos , Xenopus laevis , Animais , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Canais de Cloreto/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Feminino , Clostridium perfringens/metabolismoRESUMO
Mechanosensitive ion channels, particularly Piezo channels, are widely expressed in various tissues. However, their role in immune cells remains underexplored. Therefore, this study aimed to investigate the functional role of Piezo1 in the human eosinophil cell line AML14.3D10. We detected Piezo1 mRNA expression, but not Piezo2 expression, in these cells, confirming the presence of the Piezo1 protein. Activation of Piezo1 with Yoda1, its specific agonist, resulted in a significant calcium influx, which was inhibited by the Piezo1-specific inhibitor Dooku1, as well as other nonspecific inhibitors (Ruthenium Red, Gd3+, and GsMTx-4). Further analysis revealed that Piezo1 activation modulated the expression and secretion of both pro-inflammatory and anti-inflammatory cytokines in AML14.3D10 cells. Notably, supernatants from Piezo1-activated AML14.3D10 cells enhanced capsaicin and ATP-induced calcium responses in the dorsal root ganglion neurons of mice. These findings elucidate the physiological role of Piezo1 in AML14.3D10 cells and suggest that factors secreted by these cells can modulate the activity of transient receptor potential 1 (TRPV1) and purinergic receptors, which are associated with pain and itch signaling. The results of this study significantly advance our understanding of the function of Piezo1 channels in the immune and sensory nervous systems.
Assuntos
Eosinófilos , Canais Iônicos , Humanos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Animais , Eosinófilos/metabolismo , Eosinófilos/imunologia , Camundongos , Linhagem Celular , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Citocinas/metabolismo , Rutênio Vermelho/farmacologia , Trifosfato de Adenosina/metabolismo , Tiadiazóis/farmacologia , PirazinasRESUMO
Previously, we have demonstrated that amiodarone (AM), a widely used antiarrhythmic drug, and its major metabolite desethylamiodarone (DEA) both affect several mitochondrial processes in isolated heart and liver mitochondria. Also, we have established DEA's antitumor properties in various cancer cell lines and in a rodent metastasis model. In the present study, we compared AM's and DEA's mitochondrial and antineoplastic effects in a human triple-negative breast cancer (TNBC) cell line. Both compounds reduced viability in monolayer and sphere cultures and the invasive growth of the MDA-MB-231 TNBC line by inducing apoptosis. They lowered mitochondrial trans-membrane potential, increased Ca2+ influx, induced mitochondrial permeability transition, and promoted mitochondrial fragmentation. In accordance with their mitochondrial effects, both substances massively decreased overall, and even to a greater extent, mitochondrial ATP production decreased, as determined using a Seahorse live cell respirometer. In all these effects, DEA was more effective than AM, indicating that DEA may have higher potential in the therapy of TNBC than its parent compound.
Assuntos
Amiodarona , Antineoplásicos , Apoptose , Mitocôndrias , Neoplasias de Mama Triplo Negativas , Amiodarona/farmacologia , Amiodarona/análogos & derivados , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
Nutrient deprivation induces reserve accumulation in unicellular algae. An absence of nitrogen in the growth media results in the reorganization of the photosynthetic apparatus and triggers an increase in starch and triacylglyceride (TAG) accumulation in different algal species. Here we study the integration of photosynthetic regulatory mechanisms with carbon partitioning under N stress in C. reinhardtii. The mutant, proton gradient regulation 5 (pgr5) is impaired in photosynthetic cyclic electron flow resulting in low chloroplastic ATP/NADPH ratios. Over a time course, under both mixotrophic and phototrophic conditions, the pgr5 mutant did not accumulate starch in the first three days, but rather degraded its meagre reserves. In contrast, there was a high TAG content in the pgr5 mutant which we show, is not linked to a selective increase in autophagy in pgr5. In all strains, proteins involved in alternative electron pathways are upregulated while Photosystem II and chlorophyll are strongly degraded; pgr5 only preferentially preserved some cyt b6f complex. Our results show that low ATP/NADPH ratios due to an absence of cyclic electron flow in pgr5 result in the mobilization of starch and strong TAG accumulation from the onset of N stress in Chlamydomonas.