RESUMEN
The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1-3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.
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Benzotropina , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Inhibidores de Captación de Dopamina , Dopamina , Metilfenidato , Modelos Moleculares , Piperazinas , Humanos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Dopamina/metabolismo , Metilfenidato/farmacología , Metilfenidato/metabolismo , Metilfenidato/química , Inhibidores de Captación de Dopamina/farmacología , Inhibidores de Captación de Dopamina/metabolismo , Inhibidores de Captación de Dopamina/química , Piperazinas/farmacología , Piperazinas/metabolismo , Piperazinas/química , Benzotropina/farmacología , Benzotropina/metabolismo , Benzotropina/química , Sitios de Unión , Unión Proteica , Apoproteínas/metabolismo , Apoproteínas/química , Cocaína/farmacología , Cocaína/metabolismo , Conformación ProteicaRESUMEN
Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.
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Proteínas Munc18 , Proteínas del Tejido Nervioso , Proteínas SNARE , Membranas Sinápticas , Sintaxina 1 , Animales , Células HEK293 , Humanos , Ratones , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Dominios Proteicos , Ratas , Proteínas SNARE/química , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Membranas Sinápticas/química , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Sintaxina 1/química , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
Here, we present a novel, to the best of our knowledge, optical edge detection scheme that can be operated in both linear and circular polarization modes, leveraging an optical spatial differentiator constructed by quarter-wave Pancharatnam-Berry (P-B) phase gradient element. After explaining the theoretical mechanism, we utilize a quarter-wave P-B phase liquid crystal polarization grating to validate the dual-polarization optical edge detection capability. We demonstrate that the orientation of linear polarization and the spin of circular polarization dictate the transition between edge and bright-field images. Besides, the linear and circular polarization modes exhibit broadband and monochromatic responsive properties, respectively. This mechanism, dependent on wavelength and polarization, holds promise for applications in color image processing, chiral sensing imaging, and polarization-entangled quantum imaging.
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Lung cancer is a malignant tumor with high mortality and drug resistance. Therefore, it is urgent to explore natural and nontoxic drugs to treat lung cancer. In this study, the natural active ingredient AANL extracted from Agrocybe aegirita was used to modify nanoselenium by an oxidation-reduction method. Transmission electron microscope detection and infrared spectroscopy showed that a novel selenium nanocomposite named AANL-SeNPs was successfully prepared. The results of nanoscale characterization showed that AANL-SeNPs had good stability and uniform dispersion in aqueous solution by zeta potential and spectrum analysis. At the cellular level, we found that AANL-SeNPs significantly inhibited the cell viability of lung cancer cells, and the cell inhibition rate of 60 nM AANL-SeNPs was 39 % in H157 cells, 67 % in H147 cells, and 62 % in A549 cells. The IC50 value of AANL-SeNPs was 51.85 nM in A549 cells and 81.57 nM in H157 cells. Moreover, AANL-SeNPs could inhibit the cell proliferation and migration, and enhance the sensitivity of lung cancer cells to osimertinib and has no toxic to normal cells. In vivo, AANL-SeNPs significantly slowed tumor growth in tumor-bearing mice by establishing a subcutaneous transplantation tumor model for lung cancer, and the tumor size was smaller and was reduced about 79 % in 2 mg/kg AANL-SeNPs group compared with PBS group. Mechanistically, a total of 38 differentially expressed proteins were identified by data-independent acquisition mass spectrometry. A significantly upregulated protein, CDC-like kinase 2 (CLK2), was screened and validated for further analysis, which showed that the expression levels of CLK2 were increased in H157 and H1437 cells after AANL-SeNPs treatment. The results obtained in this study suggest that a novel selenium nanocomposite AANL-SeNPs, which inhibits lung cancer by upregulating the expression of CLK2.
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Antineoplásicos , Proliferación Celular , Neoplasias Pulmonares , Nanocompuestos , Proteínas Tirosina Quinasas , Selenio , Regulación hacia Arriba , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Nanocompuestos/química , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Animales , Selenio/química , Selenio/farmacología , Ratones , Regulación hacia Arriba/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Estructura Molecular , Relación Estructura-Actividad , Supervivencia Celular/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/metabolismo , Línea Celular Tumoral , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
An optical spatial differentiator based on the photonic spin Hall effect (PSHE) with high tunability is presented. By utilizing the characteristics of ultra-high order modes in the symmetrical metal cladding waveguide, the Fresnel reflection coefficient spectrum exhibits a narrow peak width and low trough at the resonant incident angles, resulting in high sensitivity to changes in the incident angle-induced spatial shift caused by the PSHE (the highest ∂(|r s/r p|)/∂ θ value can reach 107). After polarization transformation and extinction, the output field demonstrates differential operation with respect to the input field. When applied to edge detection, our differentiator can achieve tunable resolution edge images by adjusting the incident angle. Our proposed edge detection scheme has potential applications for cellular and molecular imaging through two-dimensional extension via the target rotation.
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Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the onset of symptoms, typically occurring later in life, and significant deficits in cognitive functions including learning, memory, speech, and behavior. Ongoing research endeavors seek to explore methods for preventing and treating AD, as well as delving into the molecular mechanisms underlying existing and novel therapeutic approaches encompassing exercise, diet, and drug regimens for individuals with AD or those at risk of developing AD. Among these interventions, dietary interventions have garnered increasing attention due to their potential in addressing the disease. Eating is among the most fundamental of human daily activities, and controlled dietary practices, such as fasting, have gained prominence as essential clinical methods for disease prevention and treatment. Research findings indicate that fasting holds promise in effectively alleviating and improving the cognitive decline associated with age or as consequence of disease. The clinical efficacy of fasting in addressing AD and related disorders might be grounded in its influence on various molecular mechanisms, including neuroinflammation, glial cell activation, insulin resistance, autophagy regulation, nerve regeneration, the gut microbiome, and accumulations of amyloid-ß and tau proteins. The present study reviews possible molecular mechanisms underpinning the therapeutic effects of fasting in patients with AD, as well as in models of the disorder, to establish a theoretical basis for using fasting as a viable approach to treat AD.
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Enfermedad de Alzheimer , Humanos , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Ayuno , Proteínas tau , Aprendizaje , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Neurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive. RESULTS: Here, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1. CONCLUSIONS: Together with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.
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Proteínas del Tejido Nervioso , Proteína 2 de Membrana Asociada a Vesículas , Ratones , Animales , Proteínas del Tejido Nervioso/genética , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Sintaxina 1/genética , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Unión ProteicaRESUMEN
Chiral transition metal oxides (TMOs) are in the forefront of research as potential active materials in various optoelectronic applications. However, the nonlinear optical (NLO) properties of the chiral TMOs have not been fully understood. Here, several kinds of copper oxide nanosheets capped with different chiral amino acids are synthesized. Notably, we investigate the NLO activities of these materials, including broadband second harmonic generation and transformation of nonlinear optical properties from saturable absorption to reverse saturable absorption. This work will broaden the use of chiral TMO materials in nonlinear photonic devices.
RESUMEN
Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.
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Actinobacteria/ultraestructura , Complejos Multiproteicos/ultraestructura , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/ultraestructura , Actinobacteria/química , Transporte Biológico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Complejo I de Transporte de Electrón/ultraestructura , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Complejos Multiproteicos/química , Oxidación-Reducción , Bombas de Protones/química , Bombas de Protones/genética , Bombas de Protones/ultraestructura , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
A hybrid optoelectronic bistability is realized with the assistance of an ultrahigh-order mode (UHM) excited in a symmetrical metal-cladding waveguide (SMCW). PMN-PT ceramics is selected as the guiding layer, which possesses the voltage modulated refractive index and thickness by means of an electro-optical effect and converse-piezoelectric effect. An amplified voltage signal translated from the intensity of reflected light is exerted on the guiding layer, whose parameter variations can alter the resonance condition of the UHM and finally lead to a dramatic change in the intensity of the reflected light. Since the full width at half-maximum of the UHM is extremely narrow, a hysteresis behavior with a milliwatt threshold between the incident light and the reflected light can be achieved when a positive feedback is established. Our bistability configuration is simple and not limited to TM polarization.
RESUMEN
A band-rejection filter is proposed based on a prism-waveguide cascaded coupling system, which is composed of an equilateral trapezium prism and a deposited multilayer structure. By properly adjusting the thickness of the coupling layer and the light extinction coefficient of the guiding layer, the radiative and intrinsic dampings matching condition could be well satisfied, and then a series of reflectivity dips will appear in the reflectivity wavelength spectrum. Since the module of reflectivity is smaller than one, the extinction ratio of the rejected frequency via the cascaded coupling system is twice as high as that of the single-coupling technology. By narrowing the guiding layer to a micrometer scale, the free spectral range is broad enough to cover the Raman spectrum scattered from the frequently used sample. In addition, the numerically calculated results show that the light in the free spectral range is mostly reflected, with an insertion loss down to 0.45 dB. Compared to previously reported band-rejection filters, it is relatively simple to manufacture our device, which possesses potential applications to help distinguish the Raman signal from the elastic scattering background.
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BACKGROUND: Heterotopic pregnancy occurred after frozen embryo transfer with two D3 embryos, and the case had a history of bilateral salpingectomy due to salpingocyesis. An ectopic heterotopic pregnancy was implanted in the left psoas major muscle, which has not been previously reported. CASE PRESENTATION: A 33-year-old woman presented with left back pain after curettage due to foetal arrest in the uterus without vaginal bleeding and spotting, and painkillers relieved the pain initially. When the painkillers ceased to work, the patient returned to the hospital. The ß-human chorionic gonadotropin (ß-hCG) level remained increased compared with the time of curettage, and a diagnosis of retroperitoneal abdominal pregnancy was suggested by ultrasonography and computerized tomography (CT) with the gestational sac implanted in the left psoas major muscle at the left hilum level. Laparotomy was performed to remove the ectopic pregnancy. During the operation, we carefully separated the adipose tissue between the space of the left kidney door and left psoas major muscle, peeled away the gestational sac that was approximately 50 mm × 40 mm with a 25-mm-long foetal bud, and gave a local injection of 10 mg of methotrexate in the psoas major muscle. Fifty days later, ß-hCG decreased to normal levels. CONCLUSION: It is necessary to pay more attention to the main complaints to exclude rare types of ectopic pregnancies of the pelvis and abdomen after embryo transfer.
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Transferencia de Embrión , Embarazo Heterotópico/diagnóstico por imagen , Adulto , Criopreservación , Femenino , Humanos , Embarazo , Tomografía Computarizada por Rayos X , Ultrasonografía PrenatalRESUMEN
Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.
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Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Transporte de Membrana/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Desoxicitidina Monofosfato , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To investigate the therapeutic effects of commonly used selective α-adrenergic receptor antagonists (α-ARA) on benign prostatic hyperplasia (BPH). METHODS: PubMed, Embase and CNKI databases were searched for the literature about selective α-ARAs for the treatment of BPH and the information was extracted on the common adverse reactions in the course of treatment. Multivariate meta-analysis was conducted to investigate the therapeutic effects of different α-ARAs. RESULTS: The total rates of adverse effects of silodosin and tamsulosin were the highest, 51.9% and 34.0% respectively, with the highest incidences of headache (38.3%), weakness (23.6%) and dizziness (17.5%). Besides, tamsulosin ranked the first in inducing sexual dysfunction of the male patients with BPH (70.4%). CONCLUSIONS: Doxazosin is preferable as the first-choice treatment of BPH for its therapeutic effect and improvement of the patient's quality of life. Silodosin and tamsulosin, however, can be selectively used according to the patient's specific tolerance to different adverse effects.
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Antagonistas Adrenérgicos alfa/uso terapéutico , Hiperplasia Prostática/tratamiento farmacológico , Antagonistas Adrenérgicos alfa/efectos adversos , Doxazosina/efectos adversos , Doxazosina/uso terapéutico , Humanos , Indoles/efectos adversos , Indoles/uso terapéutico , Masculino , Metaanálisis en Red , Calidad de Vida , Disfunciones Sexuales Fisiológicas/inducido químicamente , Tamsulosina/efectos adversos , Tamsulosina/uso terapéuticoRESUMEN
The research is aimed to study of the influence of environmental factors on the yield and quality traits, and find out the regularity of the growth and development of perilla. The main environmental factor data in six ecological area in Guizhou province were collected, and the correlation analysis with yield and quality traits of 15 perilla strains was conducted. The results showed that the cultivation environment has significant effects on the yield and quality traits of perilla. The effect of environment on main yield composed traits, contained grain number in top spike, effective panicle number per plant, plant height, top spike length, growth period, and thousand seed weight was degressive. In the different environmental factors, the latitude showed positive correlation with yield, growth period and effective panicle number per plant, and negative correlation with top spike length and grain number in top spike. Elevation showed negative correlation with the growth period of perilla. The perilla yield increased at first and then decreased with altitude rising, with the maximum in the 800 m altitude. The 600-900 m altitude is suitable area for perilla. Except for positive correlation with the plant height, and negative correlation with top spike length, the longitude showed in apparent impact on other traits. Sunshine duration, temperature and rainfall accumulation showed different effect on the different perilla strains. For yield composed traits, the sunshine duration was negatively correlation with the plant length. The accumulated temperature and mean temperature showed negative correlation with the main spike length, the rainfall showed negative correlation with the precipitation and growth period, plant height, ear number. The environmental impact on the oil compounds decreased with oleic acid, stearic acid, linoleic acid, α-linolenic acid, palmitic acid and oil content. Correlation analysis showed that the significantly negative correlation between the oil content and palmitic acid and linoleic acid content, and the positive correlation between linolenic acid content, α-linolenic acid content showed significant negative correlation with other fatty acids composition, and palmitic acid, stearic acid, oleic acid, linoleic acid showed significant positive correlation with each other. The influence of different environmental factors on the quality of perilla were as follows: the oil content was positively associated with elevation and sunshine duration. α-Linolenic acid content showed negative correlation with longitude, latitude, accumulated temperature and mean temperature, but positive correlation with altitude, sunlight and rainfall capacity. The correlation between palmitic acid, stearic acid, oleic acid, linoleic acid and environmental factors showed contrast character of α-linolenic acid. This study detailed discussed the influence of environmental factors on the quality of perilla, which provided the foundation of ecological planting technology and geoherbalism research of perilla.
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Ambiente , Perilla frutescens/química , Perilla frutescens/crecimiento & desarrollo , Fitoquímicos/análisis , Ácidos Grasos/análisis , Aceites de Plantas/análisisRESUMEN
In this paper, the tuning characteristics of locally bent microfiber taper covered with a nanosized high-refractive-index liquid crystal (LC) layer under different temperatures and electric field intensities have been theoretically analyzed and experimentally investigated. A locally bent microfiber taper interferometer with a waist diameter of â¼3.72 µm is fabricated by using the flame brushing technique, followed by bending the transition region of the taper to form a modal interferometer and later by placing a â¼200 nm LC layer over the uniform taper waist region. Experimental results indicate that a high-efficiency thermal or electric tuning of an LC-coated locally bent microfiber taper interferometer could be achieved. This suggests a potential application of this device as tunable all-fiber photonic devices, such as filters, modulators, and sensing elements.
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The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energy-coupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.
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Secuencias de Aminoácidos , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión/genética , Transporte Biológico , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ï¼Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% ß-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.
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Triticum/metabolismo , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/fisiología , Sistemas de Lectura Abierta/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
RAR1 and SGT1 are important co-chaperones of Hsp90. We previously showed that TaHsp90.1 is required for wheat seedling growth, and that TaHsp90.2 and TaHsp90.3 are essential for resistance (R) gene mediated resistance to stripe rust fungus. Here, we report the characterization of TaRAR1 and TaSGT1 genes in bread wheat. TaRAR1 and TaSGT1 each had three homoeologs, which were located on wheat groups 2 and 3 chromosomes, respectively. Strong inhibition of seedling growth was observed after silencing TaSGT1 but not TaRAR1. In contrast, decreasing the expression of TaRAR1 or TaSGT1 could all compromise R gene mediated resistance to stripe rust fungus infection. Protein-protein interactions were found among TaRAR1, TaSGT1 and TaHsp90. The N-terminus of TaHsp90, the CHORD-I and CHORD-II domains of TaRAR1 and the CS domain of TaSGT1 may be instrumental for the interactions among the three proteins. Based on this work and our previous study on TaHsp90, we speculate that the TaSGT1-TaHsp90.1 interaction is important for maintaining bread wheat seedling growth. The TaRAR1-TaSGT1-TaHsp90.2 and TaRAR1-TaSGT1-TaHsp90.3 interactions are involved in controlling the resistance to stripe rust disease. The new information obtained here should aid further functional investigations of TaRAR1-TaSGT1-TaHsp90 complexes in regulating bread wheat growth and disease resistance.
Asunto(s)
Basidiomycota/fisiología , Resistencia a la Enfermedad , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Triticum/genética , Secuencia de Bases , Silenciador del Gen , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/microbiología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/inmunología , Plantones/microbiología , Análisis de Secuencia de ADN , Triticum/crecimiento & desarrollo , Triticum/inmunología , Triticum/microbiología , Técnicas del Sistema de Dos HíbridosRESUMEN
MAIN CONCLUSION: TraeALDH7B1 - 5A , encoding aldehyde dehydrogenase 7 in wheat, conferred significant drought tolerance to Arabidopsis , supported by molecular biological and physiological experiments. Drought stress significantly affects wheat yields. Aldehyde dehydrogenase (ALDH) is a family of enzymes catalyzing the irreversible conversion of aldehydes into acids to decrease the damage caused by abiotic stresses. However, no wheat ALDH member has been functionally characterized to date. Here, we obtained a differentially expressed EST encoding ALDH7 from a cDNA-AFLP library of wheat that was treated with polyethylene glycol 6000. The three full-length homologs of TraeALDH7B1 were isolated by searching the NCBI database and by homolog-based cloning method. Using nulli-tetrasomic lines we located them on wheat chromosomes 5A, 5B and 5D, and named them as TraeALDH7B1-5A, -5B and -5D, respectively. Gene expression profiles indicated that the expressions of all three genes were induced in roots, leaves, culms and spikelets under drought and salt stresses. Enzymatic activity analysis showed that TraeALDH7B1-5A had acetaldehyde dehydrogenase activity. For further functional analysis, we developed transgenic Arabidopsis lines overexpressing TraeALDH7B1-5A driven by the cauliflower mosaic virus 35S promoter. Compared with wild type Arabidopsis, 35S::TraeALDH7B1-5A plants significantly enhanced the tolerance to drought stress, which was demonstrated by up-regulation of stress responsive genes and physiological evidence of primary root length, maintenance of water retention and contents of chlorophyll and MDA. The combined results indicated that TraeALDH7B1-5A is an important drought responsive gene for genetic transformation to improve drought tolerance in crops.