RESUMEN
Unlike natural soyasaponins and their aglycones formed by enzymatic hydrolysis in the human intestine, in vivo intermediate soyasaponin metabolites are difficult to prepare. Therefore, the pharmacological activities of in vivo intermediate soyasaponins remain uninvestigated. Herein, in vivo intermediate soyasaponins with purities of >90% were prepared by in vitro deacetylation (alkaline treatment) and deglucosylation (ß-glucosidase treatment) of natural soyasaponins using preparative high-performance liquid chromatography. These compounds exhibited higher anti-inflammatory and antioxidant activities than natural soyasaponins in in vitro bioassays, suggesting that the intermediate soyasaponins can be used as improved bioactive food supplements. To the best of our knowledge, this is the first study reporting the in vitro preparation and bioassays of in vivo intermediate soyasaponin metabolites formed in the human intestine.
RESUMEN
Compound K (C-K), one of the most bioactive ginsenoside, is produced by hydrolyzing the glycoside moieties of protopanaxadiol (PPD)-type glycosylated ginsenosides in the ginseng extract. To enhance the biotransformation of PPD-type ginsenosides in American ginseng extract (AGE) into C-K, the optimization of the feed type, concentration, and period for the carbon source sucrose and the reactant AGE was performed in fed-batch fermentation of Aspergillus tubingensis using a fermenter. The concentration (3.94 g/L) and productivity (27.4 mg/L/h) of C-K after feed optimization in fed-batch fermentation increased 3.1-fold compared to those (1.29 g/L and 8.96 mg/L/h) in batch fermentation, and a molar conversion of 100% was achieved. To the best of our knowledge, this is the first trial of fed-batch fermentation to convert ginseng extract into deglycosylated ginsenoside and the highest reported C-K concentration and productivity using ginseng extract via fermentation. After ethanol and resin treatments, C-K solids with purities of 59% and 96% were obtained from the fermentation broth as food- and pharmaceutical-grade products, respectively.
RESUMEN
Antiseizure medication is the mainstay of treatment for seizures, and adjunctive therapy is widely used to achieve adequate seizure control in patients with epilepsy who fail to respond to the first monotherapy. The newly developed antiepileptic drug cenobamate (YKP3089) as an adjunctive therapy improved seizure control in patients with uncontrolled focal seizures. Cenobamate is thought to reduce neuronal excitability through action on multiple targets, including GABA A receptors (GABAARs) and voltage-gated sodium channels. However, its effects on brain function and synaptic plasticity are unclear. Here, we explored the behavioral, synaptic, and cellular actions of cenobamate. Cenobamate influenced novel object recognition, object location memory, and Morris water maze performance in mice. Cenobamate enhanced inhibitory postsynaptic potentials by prolonging inhibitory postsynaptic current (IPSC) decay without affecting presynaptic GABA release or the peak amplitude of IPSCs. In addition, cenobamate suppressed hippocampal excitatory synaptic transmission by reducing the excitability of Schaffer collaterals and interfered with the induction of long-term potentiation. A reduction in neuronal excitability induced by cenobamate was associated with an elevation of action potential (AP) threshold, and which progressively increased in later APs during repetitive firing, indicating the activity-dependent modulation of neuronal sodium currents. Cenobamate suppressed neuronal excitability under the condition that GABAergic neurotransmission is excitatory, and administration of cenobamate rapidly enhanced the phosphorylation of eukaryotic elongation factor 2 in the hippocampus of adult and neonatal mice. Collectively, these results suggest that the combined action of cenobamate on sodium currents and GABAAR-mediated synapse responses results in reduced excitability in neurons.
Asunto(s)
Convulsiones , Transmisión Sináptica , Ratones , Animales , Convulsiones/tratamiento farmacológico , Sodio , Cognición , Ácido gamma-Aminobutírico/uso terapéuticoRESUMEN
Inborn errors of metabolism (IEMs) are common causes of neurodevelopmental disorders, including microcephaly, hyperactivity, and intellectual disability. However, the synaptic mechanisms of and pharmacological interventions for the neurological complications of most IEMs are unclear. Here, we report that metabolic dysfunction perturbs neuronal NMDA receptor (NMDAR) homeostasis and that the restoration of NMDAR signaling ameliorates neurodevelopmental and cognitive deficits in IEM model mice that lack aminopeptidase P1. Aminopeptidase P1-deficient (Xpnpep1-/-) mice, with a disruption of the proline-specific metalloprotease gene Xpnpep1, exhibit hippocampal neurodegeneration, behavioral hyperactivity, and impaired hippocampus-dependent learning. In this study, we found that GluN1 and GluN2A expression, NMDAR activity, and the NMDAR-dependent long-term potentiation (LTP) of excitatory synaptic transmission were markedly enhanced in the hippocampi of Xpnpep1-/- mice. The exaggerated NMDAR activity and NMDAR-dependent LTP were reversed by the NMDAR antagonist memantine. A single administration of memantine reversed hyperactivity in adult Xpnpep1-/- mice without improving learning and memory. Furthermore, chronic administration of memantine ameliorated hippocampal neurodegeneration, hyperactivity, and impaired learning and memory in Xpnpep1-/- mice. In addition, abnormally enhanced NMDAR-dependent LTP and NMDAR downstream signaling in the hippocampi of Xpnpep1-/- mice were reversed by chronic memantine treatment. These results suggest that the metabolic dysfunction caused by aminopeptidase P1 deficiency leads to synaptic dysfunction with excessive NMDAR activity, and the restoration of synaptic function may be a potential therapeutic strategy for the treatment of neurological complications related to IEMs.
Asunto(s)
Memantina , Receptores de N-Metil-D-Aspartato , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Animales , Hipocampo/metabolismo , Memantina/farmacología , Memantina/uso terapéutico , Ratones , N-Metilaspartato , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The ginsenoside compound K (C-K) is widely used in traditional medicines, nutritional supplements, and cosmetics owing to its diverse pharmacological activities. Although many studies on C-K production have been conducted, fermentation is reported to produce C-K with low concentration and productivity. In the present study, addition of an inducer and optimization of the carbon and nitrogen sources in the medium were performed using response surface methodology to increase the C-K production via fermentation by Aspergillus tubingensis, a generally recognized as safe fungus. The optimized inducer and carbon and nitrogen sources were 2 g/l rice straw, 10 g/l sucrose, and 10 g/l soy protein concentrate, respectively, and they resulted in a 3.1-fold increase in the concentration and productivity of C-K (0.22 g/l and 1.52 mg/l/h, respectively) compared to those used before optimization without inducer (0.071 g/l and 0.49 mg/l/h, respectively). The feeding methods of American ginseng extract (AGE), including feeding timing, feeding concentration, and feeding frequency, were also optimized. Under the optimized conditions, A. tubingensis produced 3.96 mM (2.47 g/l) C-K at 144 h by feeding two times with 8 g/l AGE at 48 and 60 h, with a productivity of 17.1 mg/l/h. The concentration and productivity of C-K after optimization of feeding methods were 11-fold higher than those before the optimization (0.22 g/l and 1.52 mg/l/h, respectively). Thus, the optimization for the feeding methods of ginseng extract is an efficient strategy to increase C-K production. To our knowledge, this is the highest reported C-K concentration and productivity via fermentation reported so far.
Asunto(s)
Panax , Aspergillus , Carbono , Fermentación , Ginsenósidos , Nitrógeno , Panax/metabolismo , Extractos Vegetales/metabolismoRESUMEN
Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABAAR) antagonist picrotoxin and the glycine receptor antagonist brucine rapidly dephosphorylated eEF2. Conversely, potentiation of GABAARs or inhibition of AMPA receptors (AMPARs) induced rapid phosphorylation of eEF2 in both the hippocampus and forebrain of mice. Chemogenetic suppression of hippocampal principal neuron activity promoted eEF2 phosphorylation. Novel context exploration and acute restraint stress rapidly modified the phosphorylation status of hippocampal eEF2. Furthermore, the hippocampal eEF2 phosphorylation levels under basal conditions were reduced in mice exhibiting epilepsy and abnormally enhanced excitability in CA3 pyramidal neurons. Collectively, the results indicated that eEF2 phosphorylation status is sensitive to neural activity and the ratio of phosphorylated eEF2 to total eEF2 could be a molecular signature for estimating neural activity in a specific brain area.
Asunto(s)
Encéfalo/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Región CA3 Hipocampal/metabolismo , Genes Reporteros , Ratones , Muscimol/farmacología , Fosforilación/efectos de los fármacos , Picrotoxina/farmacología , Prosencéfalo/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células Piramidales/metabolismo , Quinoxalinas/farmacología , Restricción Física , Estrés Fisiológico/fisiología , Estricnina/análogos & derivados , Estricnina/farmacologíaRESUMEN
Inborn errors of metabolism are often associated with neurodevelopmental disorders and brain injury. A deficiency of aminopeptidase P1, a proline-specific endopeptidase encoded by the Xpnpep1 gene, causes neurological complications in both humans and mice. In addition, aminopeptidase P1-deficient mice exhibit hippocampal neurodegeneration and impaired hippocampus-dependent learning and memory. However, the molecular and cellular changes associated with hippocampal pathology in aminopeptidase P1 deficiency are unclear. We show here that a deficiency of aminopeptidase P1 modifies the glial population and neuronal excitability in the hippocampus. Microarray and real-time quantitative reverse transcription-polymerase chain reaction analyses identified 14 differentially expressed genes (Casp1, Ccnd1, Myoc, Opalin, Aldh1a2, Aspa, Spp1, Gstm6, Serpinb1a, Pdlim1, Dsp, Tnfaip6, Slc6a20a, Slc22a2) in the Xpnpep1-/- hippocampus. In the hippocampus, aminopeptidase P1-expression signals were mainly detected in neurons. However, deficiency of aminopeptidase P1 resulted in fewer hippocampal astrocytes and increased density of microglia in the hippocampal CA3 area. In addition, Xpnpep1-/- CA3b pyramidal neurons were more excitable than wild-type neurons. These results indicate that insufficient astrocytic neuroprotection and enhanced neuronal excitability may underlie neurodegeneration and hippocampal dysfunction in aminopeptidase P1 deficiency.
Asunto(s)
Aminopeptidasas/deficiencia , Aminopeptidasas/metabolismo , Neuroglía/metabolismo , Animales , Astrocitos/metabolismo , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Hipocampo/patología , Aprendizaje/fisiología , Masculino , Memoria/fisiología , Errores Innatos del Metabolismo/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microglía/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso , Neuroglía/fisiología , Neuronas/metabolismo , Células Piramidales/metabolismoRESUMEN
SALM1 (SALM (synaptic adhesion-like molecule), also known as LRFN2 (leucine rich repeat and fibronectin type III domain containing), is a postsynaptic density (PSD)-95-interacting synaptic adhesion molecule implicated in the regulation of NMDA receptor (NMDAR) clustering largely based on in vitro data, although its in vivo functions remain unclear. Here, we found that mice lacking SALM1/LRFN2 (Lrfn2-/- mice) show a normal density of excitatory synapses but altered excitatory synaptic function, including enhanced NMDAR-dependent synaptic transmission but suppressed NMDAR-dependent synaptic plasticity in the hippocampal CA1 region. Unexpectedly, SALM1 expression was detected in both glutamatergic and GABAergic neurons and Lrfn2-/- CA1 pyramidal neurons showed decreases in the density of inhibitory synapses and the frequency of spontaneous inhibitory synaptic transmission. Behaviorally, ultrasonic vocalization was suppressed in Lrfn2-/- pups separated from their mothers and acoustic startle was enhanced, but locomotion, anxiety-like behavior, social interaction, repetitive behaviors, and learning and memory were largely normal in adult male Lrfn2-/- mice. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, and social communication and startle behaviors in mice.SIGNIFICANCE STATEMENT Synaptic adhesion molecules regulate synapse development and function, which govern neural circuit and brain functions. The SALM/LRFN (synaptic adhesion-like molecule/leucine rich repeat and fibronectin type III domain containing) family of synaptic adhesion proteins consists of five known members for which the in vivo functions are largely unknown. Here, we characterized mice lacking SALM1/LRFN2 (SALM1 KO) known to associate with NMDA receptors (NMDARs) and found that these mice showed altered NMDAR-dependent synaptic transmission and plasticity, as expected, but unexpectedly also exhibited suppressed inhibitory synapse development and synaptic transmission. Behaviorally, SALM1 KO pups showed suppressed ultrasonic vocalization upon separation from their mothers and SALM1 KO adults showed enhanced responses to loud acoustic stimuli. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, social communication, and acoustic startle behavior.
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Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Células Piramidales/fisiología , Reflejo de Sobresalto/fisiología , Vocalización Animal/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Conducta Social , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Many neuromodulating drugs acting on the nervous system originate from botanical sources. These plant-derived substances modulate the activity of receptors, ion channels, or transporters in neurons. Their properties make the substances useful for medicine and research. Here, we show that the plant lignan (+)-syringaresinol (SYR) suppresses excitatory synaptic transmission via presynaptic modulation. Bath application of SYR rapidly reduced the slopes of the field excitatory postsynaptic potentials (fEPSPs) at the hippocampal Schaffer collateral (SC)-CA1 synapse in a dose-dependent manner. SYR preferentially affected excitatory synapses, while inhibitory synaptic transmission remained unchanged. SYR had no effect on the conductance or the desensitization of AMPARs but increased the paired-pulse ratios of synaptic responses at short (20-200 ms) inter-stimulus intervals. These presynaptic changes were accompanied by a reduction of the readily releasable pool size. Pretreatment of hippocampal slices with the Gi/o protein inhibitor N-ethylmaleimide (NEM) abolished the effect of SYR on excitatory synaptic transmission, while the application of SYR significantly decreased Ca2+ currents and hyperpolarized the resting membrane potentials of hippocampal neurons. In addition, SYR suppressed picrotoxin-induced epileptiform activity in hippocampal slices. Overall, our study identifies SYR as a new neuromodulating agent and suggests that SYR suppresses excitatory synaptic transmission by modulating presynaptic transmitter release.
Asunto(s)
Estimulantes del Sistema Nervioso Central/farmacología , Furanos/farmacología , Hipocampo/efectos de los fármacos , Lignanos/farmacología , Picrotoxina/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Ácido Glutámico/farmacología , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Ratones , Técnicas de Placa-Clamp , Quinoxalinas/farmacología , Receptores AMPA/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
Antipsychotic medication is an essential component for treating schizophrenia, which is a serious mental disorder that affects approximately 1% of the global population. Olanzapine (Olz), one of the most frequently prescribed atypical antipsychotics, is generally considered a first-line drug for treating schizophrenia. In contrast to psychotic symptoms, the effects of Olz on cognitive symptoms of schizophrenia are still unclear. In addition, the mechanisms by which Olz affects the neural circuits associated with cognitive function are unknown. Here we show that Olz interrupts depotentiation (reversal of long-term potentiation) without disturbing de novo LTP (long-term potentiation) and LTD (long-term depression). At hippocampal SC-CA1 synapses, inhibition of NMDARs (N-methyl-d-aspartate receptors), mGluRs (metabotropic glutamate receptors), or mAChRs (muscarinic acetylcholine receptors) disrupted depotentiation. In addition, co-activation of NMDARs, mGluRs, and mAChRs reversed stably expressed LTP. Olz inhibits the activation of mAChRs, which amplifies glutamate signaling through enhanced NMDAR opening and Gq (Gq class of G protein)-mediated signal transduction. Behaviorally, Olz impairs spatial reversal learning of mice in the Morris water maze test. Our results uncover a novel mechanism underpinning the cognitive modulation of Olz and show that the anticholinergic property of Olz affects glutamate signaling and synaptic plasticity.
Asunto(s)
Antipsicóticos/administración & dosificación , Benzodiazepinas/administración & dosificación , Hipocampo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Aprendizaje Inverso/efectos de los fármacos , Esquizofrenia/prevención & control , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Hipocampo/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Olanzapina , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/complicaciones , Memoria Espacial/efectos de los fármacosRESUMEN
The disulfide-bond isomerase DsbC plays a crucial role in the folding of bacterial proteins in the periplasmic space. DsbC has a V-shaped dimeric structure with two domains, and Cys98 in the C-terminal domain attacks inappropriate disulfide bonds in substrate proteins due to its high nucleophilic activity. In this article, we present the crystal structure of DsbC from Salmonella enterica serovar Typhimurium. We evaluated the conserved residues Asp95 and Arg125, which are located close to Cys98. The mutation of Asp95 or Arg125 abolished the disulfide isomerase activity of DsbC in an in vitro assay using a protein substrate, and the R125A mutation significantly reduced the chaperone activity for the substrate RNase I in vivo. Furthermore, a comparative analysis suggested that the conformation of Arg125 varies depending on the packing or protein-protein interactions. Based on these findings, we suggest that Asp95 and Arg125 modulate the pKa of Cys98 during catalysis.
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Proteínas Bacterianas/química , Proteína Disulfuro Isomerasas/química , Salmonella typhimurium/enzimología , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl(2), which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H(2)O(2). SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.
Asunto(s)
Nitrobenzoatos/metabolismo , Nitrorreductasas/metabolismo , Pseudomonas fluorescens/enzimología , Secuencia de Aminoácidos , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Peróxido de Hidrógeno , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADP/metabolismo , Nitrorreductasas/genética , Oxidación-Reducción , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Recent studies in several organisms have shown that certain nascent sticky peptides stall in the ribosome during their own translation. Amino acid sequences present at the C-terminal part of Escherichia coli SecM ((150)FSTPVWISQAQGIRAGP(166)) have a well-characterized role in ribosome stalling. To investigate the determinants of the SecM motif responsible for ribosome stalling, we performed a genetic screen for mutants with an altered SecM motif that resulted in altered ribosome stalling. To do this, we used a cat fusion construct containing the SecM motif and a myc-tag (cat'-'myc-secM). This construct expresses cat'-'myc-secM mRNA transcripts predominantly translated by a subset of ribosomes called specialized ribosomes that recognize an altered ribosome binding sequence in the mRNA. While all of the isolated mutants containing mutations at the functionally conserved amino acid residues at positions between 161 and 166 showed decreased ribosome stalling, one mutant sequence containing an amino acid substitution from serine to lysine at position 157 (S157K) showed enhanced ribosome stalling that consequently increased mRNA cleavage. Our results reveal that a functionally not conserved amino acid residue at position 157 of SecM can also affect ribosome stalling and provide additional insight into the molecular mechanisms underlying sticky-peptide-induced ribosome arrest.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mutación , Biosíntesis de Proteínas , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Variación Genética , Datos de Secuencia Molecular , Ribosomas/química , Ribosomas/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
In Escherichia coli, primary rRNA transcripts must be processed by a complex process in which several ribonucleases are involved in order to generate mature 16S, 23S, and 5S rRNA molecules. While it is known that RNase G, a single-stranded RNA-specific endoribonuclease encoded by the rng gene, plays an active role in the maturation of the 5'-end of 16S rRNA, its involvement in the maturation of the 5'-end of 23S rRNA remains unclear. Here we show that E. coli cells deleted for the rng gene accumulate the 23S rRNA precursor containing an extra 77 nucleotides at its mature 5'-end. In vitro cleavage assays show that RNase G cleaves synthetic RNA containing a sequence encompassing the 5'-end to 77 nucleotides upstream of mature 23S rRNA at two sites present in single-stranded regions. Our results suggest the involvement of RNase G in the processing of the 5'-region of 23S rRNA precursors.
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Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico 23S/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Bases , Endorribonucleasas/genética , Proteínas de Escherichia coli/genética , Eliminación de Gen , Datos de Secuencia Molecular , Precursores del ARN/genética , ARN Ribosómico 23S/genéticaRESUMEN
Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.
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Escherichia coli/metabolismo , Factor 1 Procariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , ARN Ribosómico 16S/metabolismo , Ribosomas/metabolismo , Escherichia coli/genética , Mutación Puntual , Factor 1 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/genética , Factor 3 Procariótico de Iniciación/genética , ARN Ribosómico 16S/genética , Subunidades Ribosómicas/metabolismo , Ribosomas/genética , Supresión GenéticaRESUMEN
During the course of experiments aimed at identifying genes with ribonuclease III (RNase III)-dependent expression in Escherichia coli, we found that steady state levels of bdm mRNA were dependent on cellular concentrations of RNase III. The half-lives of adventitiously overexpressed bdm mRNA and the activities of a transcriptional bdm'-'cat fusion were observed to be dependent on cellular concentrations of RNase III, indicating the existence of cis-acting elements in bdm mRNA responsive to RNase III. In vitro and in vivo cleavage analyses of bdm mRNA identified two RNase III cleavage motifs, one in the 5'-untranslated region and the other in the coding region of bdm mRNA, and indicated that RNase III cleavages in the coding region constitute a rate-determining step for bdm mRNA degradation. We also discovered that downregulation of the ribonucleolytic activity of RNase III is required for the sustained elevation of RcsB-induced bdm mRNA levels during osmotic stress and that cells overexpressing bdm form biofilms more efficiently. These findings indicate that the Rcs signalling system has an additional regulatory pathway that functions to modulate bdm expression and consequently, adapt E. coli cells to osmotic stress.
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Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , ARN Mensajero/genética , Ribonucleasa III/genética , Secuencia de Bases , Cartilla de ADN , Regulación hacia Abajo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Proteínas de Escherichia coli/metabolismo , Cinética , Concentración Osmolar , Plásmidos/genética , Polimorfismo de Nucleótido Simple , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa III/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
A mutant ribosome bearing C899G in the 900 tetraloop of Escherichia coli 16S rRNA, one implicated in a conformational switch in the dynamic movements of the ribosome, showed defects in subunit association and 30S initiation complex formation. Our results explain the basis of the loss of protein synthesis ability caused by a perturbation of the 900 tetraloop.
Asunto(s)
Escherichia coli/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Pequeñas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutagénesis , Iniciación de la Cadena Peptídica Traduccional , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Ribosómico 16S/química , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Ligasas/metabolismo , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Ribosomas/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanosina Tetrafosfato/metabolismo , Ligasas/genética , Ribosomas/genética , Supresión GenéticaRESUMEN
The Streptomyces coelicolor genome harbors six copies of divergent large subunit (LSU) rRNA genes that constitute five kinds of LSU rRNA species in a cell. We report here that each heterogeneous LSU rRNA species is differentially expressed during morphological development. However, differential expression of rRNA species was not affected by depletion of a specific nutrient such as carbon, nitrogen, or phosphate from the culture medium. Analysis of the upstream region of the rRNA operons revealed that each operon contains a different composition of conserved rRNA gene promoters, indicating that each operon is independently regulated at the transcriptional level. These findings imply the existence of a regulatory mechanism that controls the independent expression of each LSU rRNA and a possible role of different species of LSU rRNA in posttranscriptional regulation of gene expression during the life cycle of this developmentally complex microorganism.
Asunto(s)
Perfilación de la Expresión Génica , ARN Ribosómico 23S/biosíntesis , ARN Ribosómico 23S/genética , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/genética , Carbono/metabolismo , ADN Bacteriano/genética , Nitrógeno/metabolismo , Fosfatos/metabolismo , Polimorfismo Genético , Regiones Promotoras GenéticasRESUMEN
The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.