Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.433
Filtrar
1.
Nat Commun ; 11(1): 5090, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037220

RESUMO

Six CO2 fixation pathways are known to operate in photoautotrophic and chemoautotrophic microorganisms. Here, we describe chemolithoautotrophic growth of the sulphate-reducing bacterium Desulfovibrio desulfuricans (strain G11) with hydrogen and sulphate as energy substrates. Genomic, transcriptomic, proteomic and metabolomic analyses reveal that D. desulfuricans assimilates CO2 via the reductive glycine pathway, a seventh CO2 fixation pathway. In this pathway, CO2 is first reduced to formate, which is reduced and condensed with a second CO2 to generate glycine. Glycine is further reduced in D. desulfuricans by glycine reductase to acetyl-P, and then to acetyl-CoA, which is condensed with another CO2 to form pyruvate. Ammonia is involved in the operation of the pathway, which is reflected in the dependence of the autotrophic growth rate on the ammonia concentration. Our study demonstrates microbial autotrophic growth fully supported by this highly ATP-efficient CO2 fixation pathway.


Assuntos
Desulfovibrio desulfuricans/crescimento & desenvolvimento , Desulfovibrio desulfuricans/metabolismo , Glicina/metabolismo , Trifosfato de Adenosina/metabolismo , Amônia/metabolismo , Processos Autotróficos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Desulfovibrio desulfuricans/genética , Perfilação da Expressão Gênica , Genoma Bacteriano , Metabolômica
3.
PLoS One ; 15(9): e0233813, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903277

RESUMO

Since the initial report of glyphosate-resistant (GR) Amaranthus palmeri S. Watson in 2006, resistant populations have been reported in 28 states. The mechanism of resistance is amplification of a 399-kb extrachromosomal circular DNA, called the EPSPS replicon, and is unique to glyphosate-resistant plants. The replicon contains a single copy of the 10-kb 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which causes the concomitant increased expression of EPSP synthase, the target enzyme of glyphosate. It is not known whether the resistance by this amplification mechanism evolved once and then spread across the country or evolved independently in several locations. To compare genomic representation and variation across the EPSPS replicon, whole genome shotgun sequencing (WGS) and mapping of sequences from both GR and susceptible (GS) biotypes to the replicon consensus sequence was performed. Sampling of GR biotypes from AZ, KS, GA, MD and DE and GS biotypes from AZ, KS and GA revealed complete contiguity and deep representation with sequences from GR plants, but lack of homogeneity and contiguity with breaks in coverage were observed with sequences from GS biotypes. The high sequence conservation among GR biotypes with very few polymorphisms which were widely distributed across the USA further supports the hypothesis that glyphosate resistance most likely originated from a single population. We show that the replicon from different populations was unique to GR plants and had similar levels of amplification.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Amaranthus/genética , Glicina/análogos & derivados , Herbicidas/metabolismo , Plantas Daninhas/genética , Amplificação de Genes , Glicina/metabolismo , Resistência a Herbicidas/genética , Proteínas de Plantas/genética , Replicon
4.
Nat Commun ; 11(1): 3752, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719334

RESUMO

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.


Assuntos
Ativação do Canal Iônico , Lipídeos/química , Nanopartículas/química , Receptores da Glicina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Glicina/metabolismo , Simulação de Dinâmica Molecular , Neurotransmissores/metabolismo , Conformação Proteica , Receptores da Glicina/ultraestrutura , Xenopus , Proteínas de Peixe-Zebra/ultraestrutura
5.
Life Sci ; 256: 117912, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504755

RESUMO

Histone deacetylase enzymes were prominent chromatin remodeling drug that targets in the pathophysiology of Alzheimer's disease associated with transcriptional dysregulation. In vitro and in vivo models of AD have demonstrated overexpression of HDAC activity. Non-specificity and non-selectivity of HDAC are the major problems of existing HDAC inhibitors. Hence, we aim to set up a methodology describing the rational development of isoform-selective HDAC inhibitor targeting class, I and class IIb. A convenient multistage virtual screening followed by machine learning and IC50 screenings were used to classify the 5064 compounds into inhibitors and non-inhibitors classes retrieved from the ChEMBL database. ADMET analysis identified the pharmacokinetics and pharmacodynamics properties of selected compounds. Molecular docking, along with mutational analysis of eleven compounds, characterized the inhibiting potency. Herein, for the first time, we reported ChEMBL1834473 (2-[[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]amino]-N-hydroxypyrimidine-5-carboxamide) as the isoform-selective HDAC inhibitor, which interact central Zn2+ atom. The negative energy and interacting residue of the ChEMBL1834473 with six HDAC isoform has also been tabulated and mapped. Moreover, our findings concluded histidine, glycine, phenylalanine, and aspartic acid as key residues in protein-ligand interaction and classify 2347 compounds as HDAC inhibitors. Later, a protein-protein interaction network of six HDAC with the key proteins involved in the progression of an AD and signaling pathway, which describes the relationship between ChEMBL1834473 and AD, has been demonstrated using PPI network where the chosen inhibitor will work. Altogether, we conclude that the compound ChEMBL1834473 may be capable of inhibiting all isoforms of class I and class IIb HDAC based on computational analysis for AD therapeutics.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Ácido Aspártico/metabolismo , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos , Glicina/metabolismo , Histona Desacetilases/metabolismo , Humanos , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Estrutura Molecular , Terapia de Alvo Molecular , Fenilalanina/metabolismo , Isoformas de Proteínas/química , Termodinâmica , Zinco/metabolismo
6.
Ecotoxicol Environ Saf ; 200: 110734, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464440

RESUMO

Microorganisms' role in pesticide degradation has been studied widely. Insitu treatments of effluents containing pesticides such as biological beds (biobeds) are efficient biological systems where biomixture (mixture of substrates) and microorganisms are the keys in pesticide treatment; however, microbial activity has been studied poorly, and its potential beyond biobeds has not been widely explored. In this study, the capacity of microbial consortium and bacteria-pure strains isolated from a biomixture (soil-straw; 1:1, v/v) used to treat agricultural effluents under real conditions were evaluated during a bioremediation process of five pesticides commonly used Yucatan Mexico. Atrazine, carbofuran, and glyphosate had the highest degradations (>90%) using the microbial consortium; 2,4-D and diazinon were the most persistent (DT50 = 8.64 and 6.63 days). From the 21 identified bacteria species in the microbial consortium, Pseudomonas nitroreducens was the most abundant (52%) according to identified sequences. For the pure strains evaluation 2,4-D (DT50 = 9.87 days), carbofuran (DT50 = 8.27 days), diazinon (DT50 = 8.80 days) and glyphosate (DT50 = 8.59 days) were less persistent in the presence of the mixed consortium (Ochrobactrum sp. DGG-1-3, Ochrobactrum sp. Ge-14, Ochrobactrum sp. B18 and Pseudomonas citronellolis strain ADA-23B). Time, pesticide, and strain type were significant (P < 0.05) in pesticide degradation, so this process is multifactorial. Microbial consortium and pure strains can be used to increase the biobed efficiency by inoculation, even in the remediation of soil contaminated by pesticides in agricultural areas.


Assuntos
Bactérias/metabolismo , Consórcios Microbianos , Praguicidas/metabolismo , Poluentes do Solo/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Agricultura , Atrazina/metabolismo , Bactérias/isolamento & purificação , Biodegradação Ambiental , Carbofurano/metabolismo , Diazinon/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Pseudomonas/isolamento & purificação , Solo/química
7.
J Neurosci ; 40(27): 5177-5195, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32457074

RESUMO

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing to profile >32,000 ACs from mice of both sexes and applied computational methods to identify 63 AC types. We identified molecular markers for each type and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two expressed glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological, and morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina.SIGNIFICANCE STATEMENT The mouse retina is a leading model for analyzing the development, structure, function, and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for the use of multiple neurotransmitters and neuropeptides, and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains ∼130 neuronal types and is therefore comparable in complexity to other regions of the brain.


Assuntos
Células Amácrinas/classificação , Retina/citologia , Células Amácrinas/metabolismo , Células Amácrinas/ultraestrutura , Animais , Feminino , Glicina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/classificação , Receptores de Neurotransmissores/metabolismo , Retina/ultraestrutura , Fator de Transcrição 4/metabolismo , Fatores de Transcrição/genética , Ácido gama-Aminobutírico/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
9.
Neuron ; 107(1): 82-94.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32330411

RESUMO

Patients lacking PYCR2, a mitochondrial enzyme that synthesizes proline, display postnatal degenerative microcephaly with hypomyelination. Here we report the crystal structure of the PYCR2 apo-enzyme and show that a novel germline p.Gly249Val mutation lies at the dimer interface and lowers its enzymatic activity. We find that knocking out Pycr2 in mice phenocopies the human disorder and depletes PYCR1 levels in neural lineages. In situ quantification of neurotransmitters in the brains of PYCR2 mutant mice and patients revealed a signature of encephalopathy driven by excessive cerebral glycine. Mechanistically, we demonstrate that loss of PYCR2 upregulates SHMT2, which is responsible for glycine synthesis. This hyperglycemia could be partially reversed by SHMT2 knockdown, which rescued the axonal beading and neurite lengths of cultured Pycr2 knockout neurons. Our findings identify the glycine metabolic pathway as a possible intervention point to alleviate the neurological symptoms of PYCR2-mutant patients.


Assuntos
Córtex Cerebral/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Glicina/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Pirrolina Carboxilato Redutases/genética , Adolescente , Animais , Córtex Cerebral/patologia , Pré-Escolar , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Linhagem , Pirrolina Carboxilato Redutases/deficiência
10.
Sci Rep ; 10(1): 3731, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111919

RESUMO

The alanine-serine-cysteine transporter Asc-1 regulates the synaptic availability of D-serine and glycine (the two co-agonists of the NMDA receptor) and is regarded as an important drug target. To shuttle the substrate from the extracellular space to the cytoplasm, this transporter undergoes multiple distinct conformational states. In this work, homology modeling, substrate docking and molecular dynamics simulations were carried out to learn more about the transition between the "outward-open" and "outward-open occluded" states. We identified a transition state involving the highly-conserved unwound TM6 region in which the Phe243 flips close to the D-serine substrate without major movements of TM6. This feature and those of other key residues are proposed to control the binding site and substrate translocation. Competitive inhibitors ACPP, LuAE00527 and SMLC were docked and their binding modes at the substrate binding site corroborated the key role played by Phe243 of TM6. For ACPP and LuAE00527, strong hydrophobic interactions with this residue hinder its mobility and prevent the uptake and the efflux of substrates. As for SMLC, the weaker interactions maintain the flexibility of Phe243 and the efflux process. Overall, we propose a molecular basis for the inhibition of substrate translocation of the Asc-1 transporter that should be valuable for rational drug design.


Assuntos
Sistema y+ de Transporte de Aminoácidos/química , Sistema y+ de Transporte de Aminoácidos/metabolismo , Motivos de Aminoácidos , Sistema y+ de Transporte de Aminoácidos/genética , Sítios de Ligação , Transporte Biológico , Glicina/química , Glicina/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Serina/química , Serina/metabolismo
11.
Biomed Res Int ; 2020: 5246350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190670

RESUMO

Purpose: To explore the effects of depression on cardiac autonomic nerve function and related metabolic pathways, the heart rate variability (HRV) and urinary differential metabolites were detected on the college students with depression. Methods: 12 female freshmen with depression were filtered by the Beck Depression Inventory (BDI-II) and Self-rating Depression Scale (SDS). By wearing an HRV monitoring system, time domain indexes and frequency domain indexes were measured over 24 hours. Liquid chromatography-mass spectrometry (LC-MS) was used to detect their urinary differential metabolites. Differential metabolites were identified by principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). The metabolic pathways related to these differential metabolites were analyzed by the MetPA database. Results: Stress time was significantly increased, and recovery time was markedly decreased in the depression group compared with the control group (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (. Conclusion: Some autonomic nervous system disruption, high stress, and poor fatigue recovery were confirmed in college students with depression. The metabolic mechanism involved the disruption of coenzyme Q biosynthesis, glycine-serine-threonine metabolism, tyrosine metabolism, pyrimidine metabolism, and steroid metabolism under daily stress.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Frequência Cardíaca/fisiologia , Ubiquinona/biossíntese , Adolescente , Depressão , Fadiga , Feminino , Glicina/metabolismo , Humanos , Metabolômica , Monitorização Fisiológica , Pirimidinas/metabolismo , Serina/metabolismo , Esteroides/metabolismo , Estresse Fisiológico , Estudantes , Treonina/metabolismo , Tirosina/metabolismo , Ubiquinona/fisiologia , Urina/química , Adulto Jovem
12.
Nat Commun ; 11(1): 1132, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111831

RESUMO

The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Glicina/metabolismo , Lisina/metabolismo , Aciltransferases/genética , Catálise , Domínio Catalítico , Coenzima A/química , Coenzima A/genética , Coenzima A/metabolismo , Cristalografia por Raios X , Humanos , Cinética , Mutação , Ácido Mirístico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
13.
Poult Sci ; 99(3): 1551-1563, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32111322

RESUMO

Reducing dietary CP can reduce N pollution. Much research has been reported in corn-based diets; however, the amino acid (AA) profiles of wheat-based diets differ. Poor performance as a result of reduced protein (RP) has been overcome in corn-based diets with essential AA and glycine (Gly) supplementation. The current study examined RP levels and Gly in wheat-based diets. An industry standard protein (SP) diet plus 3 RP diets with and without Gly supplementation, to match the SP treatment at 0.713 and 0.648% digestible Gly for the grower and finisher periods respectively, were fed to male broilers from day 10 of age. Grower CP included 22.5, 20.6, 18.3, and 17.7% (days 10-21) and finisher CP included 19.7, 17.8, 16.2, and 15.5% (days 21-35). Performance, meat yield, N efficiency, water intake, and apparent ileal digestibility of N and AA were measured. No difference in body weight gain (BWG), feed intake, or feed conversion ratio (FCR) were observed at 20% CP compared to the SP treatment. However, further reducing protein reduced BWG (P < 0.001), feed intake (P < 0.001), and increased FCR (P < 0.001). Supplementation of 0.713% Gly in the grower period increased BWG (P < 0.001) and reduced FCR (P < 0.001). Relative meat yield was not affected by dietary protein, however reducing CP increased relative fat pad weight (P < 0.001). Nitrogen efficiency increased with decreased CP in both grower (R2 = 0.69) and finisher (R2 = 0.80) treatments. Water intake decreased (R2 = 0.83) with decreasing CP intake. Apparent ileal digestibility of AA and N were higher in RP diets (P < 0.05). The benefits of reduced water intake and increased N efficiency and the disadvantages of poor performance and increased body fat in RP corn-based diets have been identified in RP wheat-based diets. Furthermore, at 18.5% CP the supplementation of crystalline AA and Gly can maintain BWG and FCR observed in SP diets.


Assuntos
Galinhas/fisiologia , Dieta com Restrição de Proteínas/veterinária , Proteínas na Dieta/análise , Digestão/efeitos dos fármacos , Ingestão de Líquidos/efeitos dos fármacos , Glicina/metabolismo , Nitrogênio/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Glicina/administração & dosagem , Íleo/fisiologia , Masculino , Carne/análise , Distribuição Aleatória , Triticum/química
14.
Int J Mol Sci ; 21(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046135

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. The early presymptomatic onset of abnormal processes is indicative of cumulative defects that ultimately lead to a late manifestation of clinical symptoms. It remains of paramount importance to identify the primary defects that underlie this condition and to determine how these deficits lead to a cycle of deterioration. We recently demonstrated that prenatal E17.5 lumbar spinal motoneurons (MNs) from SOD1G93A mice exhibit a KCC2-related alteration in chloride homeostasis, i.e., the EGABAAR is more depolarized than in WT littermates. Here, using immunohistochemistry, we found that the SOD1G93A lumbar spinal cord is less enriched with 5-HT descending fibres than the WT lumbar spinal cord. High-performance liquid chromatography confirmed the lower level of the monoamine 5-HT in the SOD1G93A spinal cord compared to the WT spinal cord. Using ex vivo perforated patch-clamp recordings of lumbar MNs coupled with pharmacology, we demonstrated that 5-HT strongly hyperpolarizes the EGABAAR by interacting with KCC2. Therefore, the deregulation of the interplay between 5-HT and KCC2 may explain the alteration in chloride homeostasis detected in prenatal SOD1G93A MNs. In conclusion, 5-HT and KCC2 are two likely key factors in the presymptomatic phase of ALS, particular in familial ALS involving the SOD1G93A mutation.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Cloretos/metabolismo , Neurônios Motores/metabolismo , Serotonina/metabolismo , Medula Espinal/metabolismo , Potenciais de Ação , Esclerose Amiotrófica Lateral/genética , Animais , Feminino , Glicina/metabolismo , Homeostase , Masculino , Camundongos , Neurônios Motores/fisiologia , Medula Espinal/embriologia , Superóxido Dismutase-1/genética , Simportadores/metabolismo , Ácido gama-Aminobutírico/metabolismo
15.
Molecules ; 25(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033280

RESUMO

The proteasome is the central component of the main cellular protein degradation pathway. During the past four decades, the critical function of the proteasome in numerous physiological processes has been revealed, and proteasome activity has been linked to various human diseases. The proteasome prevents the accumulation of misfolded proteins, controls the cell cycle, and regulates the immune response, to name a few important roles for this macromolecular "machine." As a therapeutic target, proteasome inhibitors have been approved for the treatment of multiple myeloma and mantle cell lymphoma. However, inability to sufficiently inhibit proteasome activity at tolerated doses has hampered efforts to expand the scope of proteasome inhibitor-based therapies. With emerging new modalities in myeloma, it might seem challenging to develop additional proteasome-based therapies. However, the constant development of new applications for proteasome inhibitors and deeper insights into the intricacies of protein homeostasis suggest that proteasome inhibitors might have novel therapeutic applications. Herein, we summarize the latest advances in proteasome inhibitor development and discuss the future of proteasome inhibitors and other proteasome-based therapies in combating human diseases.


Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Proteostase/efeitos dos fármacos , Compostos de Boro/metabolismo , Compostos de Boro/uso terapêutico , Bortezomib/metabolismo , Bortezomib/uso terapêutico , Glicina/análogos & derivados , Glicina/metabolismo , Glicina/uso terapêutico , Humanos , Lactonas/metabolismo , Lactonas/uso terapêutico , Oligopeptídeos/metabolismo , Oligopeptídeos/uso terapêutico , Pirróis/metabolismo , Pirróis/uso terapêutico
16.
Sci Rep ; 10(1): 3047, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080305

RESUMO

The springtail, Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.


Assuntos
Proteínas Anticongelantes/metabolismo , Artrópodes/fisiologia , Crioprotetores/metabolismo , Desidratação/metabolismo , Congelamento , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes/química , Cristalização , DNA Complementar/genética , Glicina/metabolismo , Modelos Moleculares , Isoformas de Proteínas/metabolismo
17.
Plant Mol Biol ; 103(1-2): 129-139, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32088832

RESUMO

KEY MESSAGE: The structurally simplest amino acid glycine could make contribution to nuclease activity of S-RNase and self-incompatibility in S-RNase-based plants. S-RNase is regarded as inhibitor of self-pollen tube in S-RNase-based self-incompatibility plants. Certain residues like histidine are necessary for RNase activity and self-incompatibility; however, it is unknown whether any other residues contribute to this. Previously, we identified an association between the self-compatible Chinese pear (Pyrus × bretschneideri) cultivar 'Yanzhuang' (YZ) and a mutation causing a residue shift (glycine-to-valine) in the 2nd conserved region (C2) of S21-RNase; however, it was unclear how this nonpolar aliphatic amino acid substitution caused self-compatibility. In this study, we observed that 'YZ' offspring were self-compatible when S21-RNases were all mutated. In vitro pollen tube (S21S21) growth was not completely arrested by the mutated S21-RNase. Residue frequency analysis showed that the glycine residue is highly conserved in diverse S-RNases across many plant species. We therefore generated a mutated petunia SV'-RNase (glycine to valine) and transformed it into S3LS3L petunia. The transformed pistil could not inhibit SV pollen tubes. Three-dimensional protein prediction suggested that the glycine-to-valine mutation alters the spatial structure near the active site, and RNase activity of mutated S-RNase was reducing. Thus, the glycine residue in the C2 is essential for RNase activity, substitution of this residue leads to a failure of self-incompatibility.


Assuntos
Pyrus/genética , Ribonucleases/genética , Autoincompatibilidade em Angiospermas , Domínio Catalítico , Sequência Conservada , Glicina/metabolismo , Mutação , Melhoramento Vegetal , Tubo Polínico , Pyrus/fisiologia , Ribonucleases/metabolismo , Autoincompatibilidade em Angiospermas/genética
18.
Proc Natl Acad Sci U S A ; 117(7): 3839-3847, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015122

RESUMO

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.


Assuntos
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Dimerização , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ratos , Receptores de N-Metil-D-Aspartato/genética
19.
Nat Chem Biol ; 16(5): 538-545, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32042198

RESUMO

Engineering a biotechnological microorganism for growth on one-carbon intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on one-carbon compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 h and growth yield of ~1.5 g cell dry weight (gCDW) per mol-formate. Short-term evolution decreased doubling time to less than 8 h and improved biomass yield to 2.3 gCDW per mol-formate. Growth on methanol and CO2 was achieved by further expression of a methanol dehydrogenase. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Formiatos/metabolismo , Glicina/metabolismo , Metanol/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Dióxido de Carbono/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Microrganismos Geneticamente Modificados , Mutação , Biologia Sintética/métodos
20.
Nat Chem Biol ; 16(2): 188-196, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959964

RESUMO

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.


Assuntos
Pirrolidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/metabolismo , Glicina/farmacologia , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA