RESUMO
The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multi-component protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin (CaM). This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and CaM determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.
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Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.
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Desmetilação do DNA , Osteoclastos , Animais , Diferenciação Celular/genética , Hipóxia Celular , Hipóxia/metabolismo , Camundongos , Osteoclastos/metabolismo , Oxigênio/metabolismoRESUMO
The recent discovery of TRPV6 as a pancreatitis susceptibility gene served to identify a novel mechanism of chronic pancreatitis (CP) due to Ca2+ dysregulation. Herein, we analyzed TRPV6 in 81 probands with hereditary CP (HCP), 204 probands with familial CP (FCP), and 462 patients with idiopathic CP (ICP) by targeted next-generation sequencing. We identified 25 rare nonsynonymous TRPV6 variants, 18 of which had not been previously reported. All 18 variants were characterized by a Ca2+ imaging assay, with 8 being identified as functionally deficient. Evaluation of functionally deficient variants in the three CP cohorts revealed two novel findings: (i) functionally deficient TRPV6 variants appear to occur more frequently in HCP/FCP patients than in ICP patients (3.2% vs. 1.5%) and (ii) functionally deficient TRPV6 variants found in HCP and FCP probands appear to be more frequently coinherited with known risk variants in SPINK1, CTRC, and/or CFTR than those found in ICP patients (66.7% vs 28.6%). Additionally, genetic analysis of available HCP and FCP family members revealed complex patterns of inheritance in some families. Our findings confirm that functionally deficient TRPV6 variants represent an important contributor to CP. Importantly, functionally deficient TRPV6 variants account for a significant proportion of cases of HCP/FCP.
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Canais de Cálcio , Pancreatite Crônica , Canais de Cátion TRPV , Canais de Cálcio/genética , Proteínas de Transporte/genética , Predisposição Genética para Doença , Humanos , Mutação , Pancreatite Crônica/genética , Canais de Cátion TRPV/genética , Inibidor da Tripsina Pancreática de Kazal/genéticaRESUMO
BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.
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Idade de Início , Canais de Cálcio/genética , Pancreatite Crônica/genética , Canais de Cátion TRPV/genética , Adolescente , Adulto , Idoso , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Mutação INDEL , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Pâncreas/patologia , Pancreatite Crônica/patologia , Polimorfismo de Nucleotídeo Único , Canais de Cátion TRPV/metabolismo , Sequenciamento do Exoma , Adulto JovemRESUMO
AIM: The aim of this study was to evaluate the efficacy of lurasidone in acute schizophrenia in Japan and other countries. METHODS: Subjects (aged 18-74 years) diagnosed with schizophrenia were randomized to lurasidone 40 mg/day or placebo. The primary efficacy endpoint was change from baseline on the Positive and Negative Syndrome Scale (PANSS) total score at Week 6. Secondary efficacy assessments included the Clinical Global Impression-Severity Scale (CGI-S). Safety endpoints included adverse events, and laboratory and electrocardiogram parameters. RESULTS: A total of 483 subjects were randomized to lurasidone or placebo; 107 subjects were from Japan. Mean changes from baseline at Week 6 endpoint in PANSS total scores were -19.3 in the lurasidone group and -12.7 in the placebo group (treatment difference: P < 0.001, effect size = 0.41). Changes from baseline for Week 6 CGI-S scores were -1.0 for lurasidone and -0.7 for placebo (treatment difference: P < 0.001, effect size = 0.41). All-cause discontinuation during the 6-week, double-blind period was 19.4% for lurasidone and 25.4% for placebo, and discontinuation rates due to adverse event were 5.7% for lurasidone and 6.4% for placebo. The following common treatment-emergent adverse events occurred in more than 2% on lurasidone and at a rate at least twice that of the placebo group: akathisia (4.0%), dizziness (2.8%), somnolence (2.8%), abdominal discomfort (2.0%) and asthenia (2.0%). No significant changes in bodyweight or metabolic parameters were observed. CONCLUSION: Lurasidone 40 mg once daily dosing demonstrated efficacy in a patient population with acute schizophrenia, including subjects from Japan, and was generally safe and well-tolerated.
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Terahertz (THz) irradiation has been exploited in biomedical applications involving non-invasive manipulation of living cells. We developed an apparatus for studying the effects of THz pulse irradiation on living human induced pluripotent stem cells. The THz pulse of the maximum electric field reached 0.5 MV/cm and was applied for one hour with 1 kHz repetition to the entire cell-culture area, a diameter of 1 mm. RNA sequencing of global gene-expression revealed that many THz-regulated genes were driven by zinc-finger transcription factors. Combined with a consideration of the interactions of metal ions and a THz electric field, these results imply that the local intracellular concentration of metal ions, such as Zn2+, was changed by the effective electrical force of our THz pulse.
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Redes Reguladoras de Genes/efeitos da radiação , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Radiação Terahertz , Sobrevivência Celular , Eletricidade , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição/metabolismoRESUMO
The plausible nitric oxide (NO)-sensing module of TRPC5 was incorporated in a enhanced green fluorescent protein (EGFP) to evaluate its conformational change as an optical response upon the reaction with NO. Two cysteine residues located in the NO-sensing module have been proposed to form a disulfide bond through S-nitrosylation of the thiol group by NO. Modification of the cysteine residues by NO resulted a ratiometric change of EGFP emission through transducing the conformational change of NO-sensing module to the EGFP chromophore. The oxidized form of NO-sensing module fused EGFP changed the intensity of emission spectra upon reduction of the disulfide bond at the NO-reactive module. The NO-sensing module fused EGFP in its reduced form avidly reacted with NO and realized the ratiometric fluorescence intensity changes depending on the formation of disulfide bond. These results support the notion that NO induces a conformational change at the putative NO-sensing segment of TRPC5, and provide a prototype for the genetically encoded cellular NO sensors.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Óxido Nítrico/farmacologia , Canais de Cátion TRPC/metabolismo , Escherichia coli , Proteínas de Fluorescência Verde , Humanos , Peróxido de Hidrogênio , Imagem Óptica , Relação Estrutura-Atividade , Canais de Cátion TRPC/químicaRESUMO
BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.
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Cálcio/metabolismo , Calmodulina/metabolismo , Citoesqueleto/ultraestrutura , Glomerulosclerose Segmentar e Focal/genética , Canal de Cátion TRPC6/genética , Actinas/ultraestrutura , Animais , Sítios de Ligação , Calmodulina/genética , Mutação com Ganho de Função , Glomerulosclerose Segmentar e Focal/metabolismo , Células HEK293 , Humanos , Camundongos , Fenótipo , Podócitos , Domínios Proteicos , Canal de Cátion TRPC6/ultraestruturaRESUMO
Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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Aptâmeros de Nucleotídeos/química , Biossíntese de Proteínas , Sondas RNA/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Compostos de Benzil/química , Escherichia coli/genética , Corantes Fluorescentes , Imidazolinas/química , Microscopia de Fluorescência , Ribossomos/metabolismo , Spinacia oleracea/genéticaRESUMO
Temperature influences the activities of living organisms at various levels. Cells not only detect environmental temperature changes through their unique temperature-sensitive molecular machineries but also muster an appropriate response to the temperature change to maintain their inherent functions. Despite the fundamental involvement of temperature in physiological phenomena, the mechanism by which cells produce and use heat is largely unknown. Recently, fluorescent thermosensors that function as thermometers in live cells have attracted much attention in biology. These new tools, made of various temperature-sensitive molecules, have allowed for intracellular thermometry at the single-cell level. Intriguing spatiotemporal temperature variations, including organelle-specific thermogenesis, have been revealed with these fluorescent thermosensors, which suggest an intrinsic connection between temperature and cell functions. Moreover, fluorescent thermosensors have shown that intracellular temperature changes at the microscopic level are largely different from those assumed for a water environment at the macroscopic level. Thus, the employment of fluorescent thermosensors will uncover novel mechanisms of intracellular temperature-assisted physiological functions.
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Técnicas Biossensoriais/métodos , Organelas/metabolismo , Termometria/métodos , Xantenos/química , Animais , Proteínas de Fluorescência Verde/química , Humanos , Pontos Quânticos/químicaRESUMO
In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.
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Proteínas de Fluorescência Verde/química , Salmonella enterica/metabolismo , Temperatura , Adenoviridae/genética , Adipócitos Marrons/citologia , Proteínas de Bactérias/química , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Células HeLa , Temperatura Alta , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Conformação ProteicaRESUMO
Plasmodium parasites possess two endosymbiotic organelles: a mitochondrion and a relict plastid called the apicoplast. To accommodate the translational requirements of these organelles in addition to its cytosolic translation apparatus, the parasite must maintain a supply of charged tRNA molecules in each of these compartments. In the present study we investigate how the parasite manages these translational requirements for charged tRNACys with only a single gene for CysRS (cysteinyl-tRNA synthetase). We demonstrate that the single PfCysRS (Plasmodium falciparum CysRS) transcript is alternatively spliced, and, using a combination of endogenous and heterologous tagging experiments in both P. falciparum and Toxoplasma gondii, we show that CysRS isoforms traffic to the cytosol and apicoplast. PfCysRS can recognize and charge the eukaryotic tRNACys encoded by the Plasmodium nucleus as well as the bacterial-type tRNA encoded by the apicoplast genome, albeit with a preference for the eukaryotic type cytosolic tRNA. The results of the present study indicate that apicomplexan parasites have lost their original plastidic cysteinyl-tRNA synthetase, and have replaced it with a dual-targeted eukaryotic type CysRS that recognizes plastid and nuclear tRNACys. Inhibitors of the Plasmodium dual-targeted CysRS would potentially offer a therapy capable of the desirable immediate effects on parasite growth as well as the irreversibility of inhibitors that disrupt apicoplast inheritance.
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Aminoacil-tRNA Sintetases/metabolismo , Citosol/metabolismo , Plasmodium falciparum/enzimologia , Processamento Alternativo , Aminoacil-tRNA Sintetases/genética , Apicoplastos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutação , Plasmodium falciparum/genética , Transporte Proteico , Temperatura , Toxoplasma/genéticaRESUMO
Charcot-Marie-Tooth disease type 2D (CMT2D) is an autosomal-dominant axonal peripheral neuropathy characterized by impaired motor and sensory function in the distal extremities. Mutations in the glycyl-tRNA synthetase (GARS) gene cause CMT2D. GARS is a member of the ubiquitously expressed aminoacyl-tRNA synthetase (ARS) family and is responsible for charging tRNA with glycine. To date, 13 GARS mutations have been identified in patients with CMT disease. While functional studies have revealed loss-of-function characteristics, only four GARS mutations have been rigorously studied. Here, we report the functional evaluation of nine CMT-associated GARS mutations in tRNA charging, yeast complementation, and subcellular localization assays. Our results demonstrate that impaired function is a common characteristic of CMT-associated GARS mutations. Additionally, one mutation previously associated with CMT disease (p.Ser581Leu) does not demonstrate impaired function, was identified in the general population, and failed to segregate with disease in two newly identified families with CMT disease. Thus, we propose that this variant is not a disease-causing mutation. Together, our data indicate that impaired function is a key component of GARS-mediated CMT disease and emphasize the need for careful genetic and functional evaluation before implicating a variant in disease onset.
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Estudos de Associação Genética , Glicina-tRNA Ligase/genética , Glicina-tRNA Ligase/metabolismo , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Sequência de Aminoácidos , Aminoacilação , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Sequência Conservada , Análise Mutacional de DNA , Feminino , Expressão Gênica , Glicina-tRNA Ligase/química , Humanos , Cinética , Masculino , Camundongos , Neurônios/metabolismo , Linhagem , Transporte Proteico , Leveduras/genética , Leveduras/metabolismoRESUMO
Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.
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Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Mutação/genética , RNA de Transferência/metabolismo , Temperatura , tRNA Metiltransferases/genética , Alelos , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluorescência , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Desnaturação Proteica , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismoRESUMO
A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.
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Proteínas de Bactérias/química , RNA Bacteriano/química , RNA de Transferência/química , Thermus thermophilus/química , tRNA Metiltransferases/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estrutura Terciária de Proteína , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismoRESUMO
Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.
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Guanosina/metabolismo , tRNA Metiltransferases/metabolismo , Sequência de Bases , Guanosina/química , Cinética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/químicaRESUMO
Purpose: To evaluate the effect of lurasidone on a new, patient Life Engagement scale in schizophrenia. Patients and Methods: This post-hoc analysis included participants (ages 18 to 74) diagnosed with schizophrenia who were randomized to lurasidone (40 mg/day) or placebo in a 6-week double-blind efficacy study and those who continued in a subsequent 12-week open-label extension study during which patients received either 40 or 80/mg day lurasidone (flexibly dosed). Change in life engagement was measured using the Positive and Negative Syndrome Scale (PANSS) 11-item Life Engagement subscale score, and individual subscale items, at week 6 during the double-blind phase and extension phase week 12 during the open-label extension phase. Results: Analysis focused on 478 subjects randomized to lurasidone or placebo during the 6-week trial, and 146 who received lurasidone during the extension phase. During the 6-week trial, there was a significantly greater change on the PANSS11 Life Engagement subscale score from baseline to week 6 in the lurasidone group compared to the placebo group (mean changes of -6.4 and -4.8, respectively, p = 0.006; effect size = 0.27). Further improvement was evident during the extension phase for patients who received lurasidone in both phases, with a mean change from double-blind baseline to week 12 of the open-label treatment phase of -10.1 on in PANSS11 Life Engagement subscale. Conclusion: This post-hoc analysis suggests that lurasidone may improve life engagement in patients with schizophrenia, a meaningful outcome from patients' perspective. Further studies are needed to confirm this effect. Eudract Number: Trial registration: EudraCT Numbers: 2016-000060-42; 2016-000061-23.
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Objective: To evaluate the effects of lurasidone on social functioning in schizophrenia over the course of a 6-week, double-blind, placebo-controlled study and a subsequent 12-week open-label extension study.Methods: A total of 478 patients with schizophrenia (per DSM-IV-TR criteria) randomized to either lurasidone 40 mg/d (n = 245) or placebo (n = 233) in the initial 6-week double-blind study (initiated May 2016, completed November 2018) were included in the analysis. Longer-term changes were examined in a sample of 146 patients who received lurasidone, and 141 who received placebo, during the 6-week study and received flexibly dosed (40-80 mg/d) lurasidone during the 12-week extension phase. The 4-item Positive and Negative Syndrome Scale (PANSS) prosocial subscale was used to examine changes in social functioning.Results: At week 6 of the double-blind phase, lurasidone-treated patients had significantly greater improvement on the PANSS prosocial subscale compared to placebo-treated patients (P < .01, effect size at week 6 = 0.33). Significant differences from placebo were also evident at week 2 (P < .05), week 4 (P < .001), and week 5 (P < .01). Across the 12-week extension phase, patients who received lurasidone during both the 6-week double-blind phase and the 12-week open-label phase continued to show successive decreases in scores on the 4-item PANSS prosocial subscale (score change of -3.0 from double-blind baseline to week 6; mean score change of -4.2 from double-blind baseline to week 12 of the extension phase).Conclusions: In patients with schizophrenia treated with lurasidone, social functioning improved relative to placebo during a 6-week double-blind study and continued to improve over the course of 12 weeks of extension treatment with lurasidone. Effects of lurasidone on social functioning appear to be comparable to what has been reported for other atypical antipsychotics.Trial Registration: EudraCT Numbers: 2016-000060-42 and 2016-000061-23.
Assuntos
Antipsicóticos , Esquizofrenia , Humanos , Cloridrato de Lurasidona/efeitos adversos , Esquizofrenia/tratamento farmacológico , Esquizofrenia/induzido quimicamente , Interação Social , Antipsicóticos/efeitos adversos , Tempo , Método Duplo-Cego , Resultado do TratamentoRESUMO
A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.
Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais , Corantes Fluorescentes/química , Guanosina Trifosfato/análise , Peptídeos/química , Técnicas Biossensoriais/instrumentação , Modelos MolecularesRESUMO
Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.