RESUMO
Prematurely-born infants cared for in the neonatal units suffer from memory and learning deficits. Prematurity diminishes neurogenesis and synaptogenesis in the hippocampal dentate gyrus (DG). This dysmaturation of neurons is attributed to elevated PSD95, NMDR2A, and IGF1 levels. Since oestrogen treatment plays key roles in the development and plasticity of DG, we hypothesized that 17ß-estradiol (E2) treatment would ameliorate neurogenesis and synaptogenesis in the DG, reversing cognitive deficits in premature newborns. Additionally, E2-induced recovery would be mediated by IGF1 signalling. These hypotheses were tested in a rabbit model of prematurity and nonmaternal care, in which premature kits were gavage-fed and reared by laboratory personnel. We compared E2- and vehicle-treated preterm kits for morphological, molecular, and behavioural parameters. We also treated kits with oestrogen degrader, RAD1901, and assessed IGF1 signalling. We found that E2 treatment increased the number of Tbr2+ and DCX+ neuronal progenitors and increased the density of glutamatergic synapses in the DG. E2 treatment restored PSD95 and NMDAR2A levels and cognitive function in preterm kits. Transcriptomic analyses showed that E2 treatment contributed to recovery by influencing interactions between IGF1R and neurodegenerative, as well as glutamatergic genes. ERα expression was reduced on completion of E2 treatment at D7, followed by D30 elevation. E2-induced fluctuation in ERα levels was associated with a reciprocal elevation in IGF1/2 expression at D7 and reduction at D30. ERα degradation by RAD1901 treatment enhanced IGF1 levels, suggesting ERα inhibits IGF1 expression. E2 treatment alleviates the prematurity-induced maldevelopment of DG and cognitive dysfunctions by regulating ERα and IGF1 levels.
Assuntos
Receptor alfa de Estrogênio , Estrogênios , Animais , Coelhos , Tetra-Hidronaftalenos , Receptores de Estrogênio , Proteína 4 Homóloga a Disks-Large/genética , Giro DenteadoRESUMO
Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.
Assuntos
Disfunção Cognitiva , Insulinas , Animais , Coelhos , Giro Denteado/metabolismo , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Fatores de Transcrição/metabolismo , Cognição , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Insulinas/metabolismoRESUMO
Premature infants with germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) suffer from neurobehavioural deficits as they enter childhood and adolescence. Yet the underlying mechanisms remain unclear. Impaired development and function of interneurons contribute to neuropsychiatric disorders. Therefore, we hypothesized that the occurrence of IVH would reduce interneuron neurogenesis in the medial ganglionic eminence and diminish the population of parvalbumin+ and somatostatin+ cortical interneurons. Because Sonic Hedgehog promotes the production of cortical interneurons, we also postulated that the activation of Sonic Hedgehog signalling might restore neurogenesis, cortical interneuron population, and neurobehavioural function in premature newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH and autopsy samples from human preterm infants. We compared premature newborns with and without IVH for intraneuronal progenitors, cortical interneurons, transcription factors regulating neurogenesis, single-cell transcriptome of medial ganglionic eminence and neurobehavioural functions. We treated premature rabbit kits with adenovirus expressing Sonic Hedgehog (Ad-Shh) or green fluorescence protein gene to determine the effect of Sonic Hedgehog activation on the interneuron production, cortical interneuron population and neurobehaviour. We discovered that IVH reduced the number of Nkx2.1+ and Dlx2+ progenitors in the medial ganglionic eminence of both humans and rabbits by attenuating their proliferation and inducing apoptosis. Moreover, IVH decreased the population of parvalbumin+ and somatostatin+ neurons in the frontal cortex of both preterm infants and kits relative to controls. Sonic Hedgehog expression and the downstream transcription factors, including Nkx2.1, Mash1, Lhx6 and Sox6, were also reduced in kits with IVH. Consistent with these findings, single-cell transcriptomic analyses of medial ganglionic eminence identified a distinct subpopulation of cells exhibiting perturbation in genes regulating neurogenesis, ciliogenesis, mitochondrial function and MAPK signalling in rabbits with IVH. More importantly, restoration of Sonic Hedgehog level by Ad-Shh treatment ameliorated neurogenesis, cortical interneuron population and neurobehavioural function in kits with IVH. Additionally, Sonic Hedgehog activation alleviated IVH-induced inflammation and several transcriptomic changes in the medial ganglionic eminence. Taken together, IVH reduced intraneuronal production and cortical interneuron population by downregulating Sonic Hedgehog signalling in both preterm rabbits and humans. Notably, activation of Sonic Hedgehog signalling restored interneuron neurogenesis, cortical interneurons and cognitive function in rabbit kits with IVH. These findings highlight disruption in cortical interneurons in IVH and identify a novel therapeutic strategy to restore cortical interneurons and cognitive function in infants with IVH. These studies can accelerate the development of new therapies to enhance the neurodevelopmental outcome of survivors with IVH.
Assuntos
Proteínas Hedgehog , Parvalbuminas , Animais , Recém-Nascido , Humanos , Coelhos , Criança , Proteínas Hedgehog/metabolismo , Parvalbuminas/metabolismo , Parvalbuminas/farmacologia , Recém-Nascido Prematuro , Fatores de Transcrição/genética , Cognição , Hemorragia , Interneurônios/metabolismo , Somatostatina/metabolismo , Somatostatina/farmacologiaRESUMO
Intraventricular hemorrhage (IVH) remains a major complication of prematurity, worldwide. The severity of IVH is variable, ranging from a tiny germinal matrix bleed to a moderate-to-large ventricular hemorrhage or periventricular hemorrhagic infarction. Survivors with IVH often suffer from hydrocephalus and white matter injury. There is no tangible treatment to prevent post-hemorrhagic cerebral palsy, cognitive deficits, or hydrocephalus in these infants. White matter injury is attributed to blood-induced damage to axons and maturing oligodendrocyte precursors, resulting in reduced myelination and axonal loss. Hydrocephalus results from obstructed CSF circulation by blood clots, increased CSF production, and reduced CSF absorption by lymphatics and arachnoid villi. Several strategies to promote neurological recovery have shown promise in animal models, including the elimination of blood and blood products, alleviating cerebral inflammation and oxidative stress, as well as promoting survival and maturation of oligodendrocyte precursors. The present review integrates novel mechanisms of brain injury in IVH and the imminent therapies to alleviate post-hemorrhagic white matter injury and hydrocephalus in the survivors with IVH.
Assuntos
Lesões Encefálicas , Hidrocefalia , Doenças do Prematuro , Animais , Encéfalo , Lesões Encefálicas/complicações , Hemorragia Cerebral/complicações , Ventrículos Cerebrais , Humanos , Hidrocefalia/complicações , Recém-NascidoRESUMO
Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.
Assuntos
Hemorragia Cerebral Intraventricular/metabolismo , Proteínas da Mielina/biossíntese , PPAR gama/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Animais Recém-Nascidos , Antiporters/deficiência , Antiporters/metabolismo , Hemorragia Cerebral Intraventricular/patologia , Modelos Animais de Doenças , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Recém-Nascido Prematuro , Microglia/metabolismo , Doenças Mitocondriais/metabolismo , Oligodendroglia/patologia , PPAR gama/agonistas , Transtornos Psicomotores/metabolismo , Coelhos , Rosiglitazona/farmacologia , Análise de Sequência de RNA/métodosRESUMO
Preterm-born children suffer from neurological and behavioral disorders. Herein, we hypothesized that premature birth and non-maternal care of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and dendritic spine density. Additionally, we assessed whether 17ß-estradiol (E2) replacement or the TrkB receptor agonist, 7,8-dihydroxyflavone (DHF), would reverse compromised dendritic development and cognitive function in preterm newborns. These hypotheses were tested by comparing preterm (E28.5) rabbit kits cared and gavage-fed by laboratory personnel and term-kits reared and breast-fed by their mother doe at an equivalent postconceptional age. Neurobehavioral tests showed that both premature-birth and formula-feeding with non-maternal care led to increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term-kits. Dendritic branching and number of total or mushroom dendritic spines were reduced in the CA1 field of preterm-kits compared with term controls. While CDC42 and Rac1/2/3 expression levels were lower, RhoA-activity was higher in preterm-kits compared with term controls. Both E2 and DHF treatment reversed prematurity-induced reduction in spine density, reduced total RhoA-GTPase levels, and enhanced cognitive function. Hence, prematurity and non-maternal care result in cognitive deficits, and reduced dendritic arbors and spines in CA1. E2 replacement or DHF treatment might reverse changes in dendritic spines and improve neurodevelopment in premature infants.
Assuntos
Cognição/fisiologia , Espinhas Dendríticas/patologia , Estradiol/farmacologia , Hipocampo/patologia , Nascimento Prematuro/fisiopatologia , Receptor trkB/agonistas , Animais , Cognição/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Flavonas/farmacologia , Hipocampo/efeitos dos fármacos , Privação Materna , Gravidez , Nascimento Prematuro/patologia , Coelhos , Receptor trkB/efeitos dos fármacosRESUMO
Development of cortical interneurons continues until the end of human pregnancy. Premature birth deprives the newborns from the supply of maternal estrogen and a secure intrauterine environment. Indeed, preterm infants suffer from neurobehavioral disorders. This can result from both preterm birth and associated postnatal complications, which might disrupt recruitment and maturation of cortical interneurons. We hypothesized that interneuron subtypes, including parvalbumin-positive (PV+), somatostatin-positive (SST+), calretinin-positive (CalR+), and neuropeptide Y-positive (NPY+) interneurons, were recruited in the upper and lower cortical layers in a distinct manner with advancing gestational age. In addition, preterm birth would disrupt the heterogeneity of cortical interneurons, which might be reversed by estrogen treatment. These hypotheses were tested by analyzing autopsy samples from premature infants and evaluating the effect of estrogen supplementation in prematurely delivered rabbits. The PV+ and CalR+ neurons were abundant, whereas SST+ and NPY+ neurons were few in cortical layers of preterm human infants. Premature birth of infants reduced the density of PV+ or GAD67+ neurons and increased SST+ interneurons in the upper cortical layers. Importantly, 17 ß-estradiol treatment in preterm rabbits increased the number of PV+ neurons in the upper cortical layers relative to controls at postnatal day 14 (P14) and P21 and transiently reduced SST population at P14. Moreover, protein and mRNA levels of Arx, a key regulator of cortical interneuron maturation and migration, were higher in estrogen-treated rabbits relative to controls. Therefore, deficits in PV+ and excess of SST+ neurons in premature newborns are ameliorated by estrogen replacement, which can be attributed to elevated Arx levels. Estrogen replacement might enhance neurodevelopmental outcomes in extremely preterm infants.SIGNIFICANCE STATEMENT Premature birth often leads to neurodevelopmental delays and behavioral disorders, which may be ascribed to disturbances in the development and maturation of cortical interneurons. Here, we show that preterm birth in humans is associated with reduced population of parvalbumin-positive (PV+) neurons and an excess of somatostatin-expressing interneurons in the cerebral cortex. More importantly, 17 ß-estradiol treatment increased the number of PV+ neurons in preterm-born rabbits, which appears to be mediated by an elevation in the expression of Arx transcription factor. Hence the present study highlights prematurity-induced reduction in PV+ neurons in human infants and reversal in their population by estrogen replacement in preterm rabbits. Because preterm birth drops plasma estrogen level 100-fold, estrogen replacement in extremely preterm infants might improve their developmental outcome and minimize neurobehavioral disorders.
Assuntos
Córtex Cerebral/patologia , Estradiol/farmacologia , Doenças do Prematuro/patologia , Interneurônios/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Calbindina 2/análise , Contagem de Células , Feminino , Idade Gestacional , Glutamato Descarboxilase/análise , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Interneurônios/química , Interneurônios/classificação , Interneurônios/fisiologia , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neuropeptídeo Y/análise , Parvalbuminas/análise , Coelhos , Somatostatina/análise , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Intraventricular hemorrhage (IVH) in preterm infants results in reduced proliferation and maturation of oligodendrocyte progenitor cells (OPCs), and survivors exhibit reduced myelination and neurological deficits. Wnt signaling regulates OPC maturation and myelination in a context dependent manner. Herein, we hypothesized that the occurrence of IVH would downregulate Wnt signaling, and that activating Wnt signaling by GSK-3ß inhibition or Wnt3A recombinant human protein (rh-Wnt3A) treatment might promote maturation of OPCs, myelination of the white matter, and neurological recovery in premature rabbits with IVH. These hypotheses were tested in autopsy samples from preterm infants and in a rabbit model of IVH. Induction of IVH reduced expressions of activated ß-catenin, TCF-4, and Axin2 transcription factors in preterm newborns. Both AR-A014418 (ARA) and Wnt-3A treatment activated Wnt signaling. GSK-3ß inhibition by intramuscular ARA treatment accelerated maturation of OPCs, myelination, and neurological recovery in preterm rabbits with IVH compared to vehicle controls. In contrast, intracerebroventricular rh-Wnt3A treatment failed to enhance myelination and neurological function in rabbits with IVH. ARA treatment reduced microglia infiltration and IL1ß expression in rabbits with IVH relative to controls, whereas Wnt3A treatment elevated TNFα, IL1ß, and IL6 expression without affecting microglia density. GSK-3ß inhibition downregulated, while rh-Wnt3A treatment upregulated Notch signaling; and none of the two treatments affected the Sonic-Hedgehog pathway. The administration of ARA or rh-Wnt3A did not affect gliosis. The data suggest that GSK-3ß inhibition promoted myelination by suppressing inflammation and Notch signaling; and Wnt3A treatment failed to enhance myelination because of its pro-inflammatory activity and synergy with Notch signaling. GSK-3ß inhibitors might improve the neurological outcome of preterm infants with IVH.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/biossíntese , Recém-Nascido Prematuro/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Proteína Wnt3A/biossíntese , Animais , Encéfalo/efeitos dos fármacos , Feminino , Humanos , Recém-Nascido , Masculino , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Coelhos , Proteínas Recombinantes/biossíntese , Tiazóis/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1+, Dlx2+, and Sox2+ cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1+ progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2+ cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1+ and Dlx2+ cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants.SIGNIFICANCE STATEMENT Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants.
Assuntos
Animais Recém-Nascidos/fisiologia , Estrogênios/uso terapêutico , Interneurônios/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Feminino , Gânglios/citologia , Gânglios/crescimento & desenvolvimento , Gânglios/metabolismo , Proteínas de Homeodomínio/metabolismo , Hipóxia Encefálica/induzido quimicamente , Hipóxia Encefálica/patologia , Antígeno Ki-67/metabolismo , Células-Tronco Neurais/metabolismo , Gravidez , Nascimento Prematuro , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Coelhos , Fatores de Transcrição SOXB1/metabolismo , Fator Nuclear 1 de Tireoide/metabolismoRESUMO
Bacterial Topoisomerase I is a potential target for the identification of novel topoisomerase poison inhibitors that could provide leads for a new class of antibacterial compounds. Here we describe in detail a fluorescence-based cleavage assay that is successfully used in HTS for the discovery of bacterial topoisomerase Ι poisons.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Inibidores da Topoisomerase I/síntese química , Yersinia pestis/enzimologia , DNA Bacteriano/química , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Fluorescência , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Yersinia pestis/efeitos dos fármacosRESUMO
Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Topoisomerases Tipo I/química , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Lisina/metabolismo , Nuclease do Micrococo/metabolismo , Oligonucleotídeos/metabolismo , Oxirredução , Peptídeos/química , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52Å resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel ß-sheet stabilized by a crossing-over α-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA Topoisomerases Tipo I/genética , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Conformação Proteica , Alinhamento de SequênciaRESUMO
Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain and zinc ribbon-like domain bind ssDNA with primarily π-stacking interactions. This novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3ß from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.
Assuntos
DNA Topoisomerases Tipo I/química , DNA de Cadeia Simples/química , DNA Super-Helicoidal/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response.
Assuntos
Antibacterianos/farmacologia , DNA Topoisomerases Tipo I/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Mutação da Fase de Leitura , Regulação Bacteriana da Expressão Gênica , DNA Topoisomerases Tipo I/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Testes de Sensibilidade Microbiana , Taxa de Mutação , Mycobacterium tuberculosis/química , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Quinolonas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rifampina/farmacologia , Resposta SOS em Genética/efeitos dos fármacos , Elongação da Transcrição Genética , Trimetoprima/farmacologiaRESUMO
Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3ß may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Mercúrio/farmacologia , Compostos Organomercúricos/farmacologia , Inibidores da Topoisomerase I/farmacologia , Zinco/metabolismo , Cisteína/metabolismo , DNA Topoisomerases Tipo I/química , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligação ProteicaRESUMO
To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg(2+). In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg(2+) on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg(2+) coordinated EctopoI (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg(2+)EctopoI-pBAD/Thio interactions (~0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (~0.017 s(-1)). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg(2+) and furthers the understanding of importance of the Mg(2+) ion for bacterial topoisomerase I catalytic activity.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Plasmídeos/metabolismo , Magnésio/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds.
Assuntos
Antibacterianos/farmacologia , Ascomicetos/química , DNA Topoisomerases Tipo I/metabolismo , Depsídeos/farmacologia , Hidroxibenzoatos/farmacologia , Inibidores da Topoisomerase/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/toxicidade , Produtos Biológicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Depsídeos/isolamento & purificação , Depsídeos/toxicidade , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ensaios de Triagem em Larga Escala , Humanos , Hidroxibenzoatos/isolamento & purificação , Hidroxibenzoatos/toxicidade , Magnésio/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores da Topoisomerase/isolamento & purificação , Inibidores da Topoisomerase/toxicidade , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/enzimologiaRESUMO
Escherichia coli topoisomerase I (EcTopoI) is a type IA bacterial topoisomerase which is receiving large attention due to its potential application as novel target for antibacterial therapeutics. Nevertheless, a detailed knowledge of its mechanism of action at molecular level is to some extent lacking. This is partly due to the requirement of several factors (metal ions, nucleic acid) to the proper progress of the enzyme catalytic cycle. Additionally, each of them can differently affect the protein structure. Here we assess the role of the different components (DNA, metal ions, protein domains) in a dynamic environment as in solution by monitoring the catalytic as well as the structural properties of EcTopoI. Our results clearly indicated the interaction among these components as functionally relevant and underlined their mutual involvement. Some similarities with other enzymes of the same family emerged (for example DNA prevents divalent metal ions coordination at non selective binding sites). Interestingly, same interactions (C- and N-terminal domain interaction) appear to be peculiar of this bacterial topoisomerase which suggest they could be favorably exploited to the design of selective inhibitors for this class of enzyme.
Assuntos
DNA Topoisomerases Tipo I/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Magnésio/química , Manganês/química , Mapeamento de Interação de Proteínas/métodos , Sítios de Ligação , Cálcio/química , Dicroísmo Circular , Clivagem do DNA , DNA Bacteriano/química , Ativação Enzimática , Ensaios Enzimáticos , Íons/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Soluções/química , Fatores de TempoRESUMO
OBJECTIVES: Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level. METHODS: In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage-religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays. RESULTS: DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage-religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I. CONCLUSIONS: This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.