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1.
Nat Commun ; 12(1): 4621, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330928

RESUMO

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato
2.
J Med Chem ; 64(13): 9078-9099, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34129329

RESUMO

Fibroblast growth factor receptors (FGFRs) have become promising therapeutic targets in various types of cancers. In fact, several selective irreversible inhibitors capable of covalently reacting with the conserved cysteine of FGFRs are currently being evaluated in clinical trials. In this article, we optimized and discovered a novel lead compound 36 with remarkable inhibitory effects against FGFR (1-3), which is a derivative of 2H-pyrazolo[3,4-d]pyrimidine. The irreversible binding to FGFRs was characterized by LC-MS. This compound has been shown to exhibit significant anti-proliferation effects against NCI-H1581 and SNU-16 cancer cell lines both in vitro and in vivo. Compound 36 has also demonstrated a low toxicity profile and adequate pharmacokinetic properties and is currently under validation as a potential drug candidate.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
3.
Proc Natl Acad Sci U S A ; 118(16)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33853951

RESUMO

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

4.
J Colloid Interface Sci ; 596: 468-478, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848749

RESUMO

Mesocrystals are types of fascinating multifunctional materials in fabricating rapid charge transport pathways, and surface engineering could be considered as a significant influencing factor in boosting charge separation for efficient photocatalytic application. In this work, surface engineered Ta2O5-x mesocrystals were synthesized by facile alkali treatment strategy for enhanced visible light photocatalytic tetracycline degradation. The highly enhanced photocatalytic activity could be attributed to the highly increased surface areas and surface hydroxyl groups to compare with those of commercial Ta2O5 and pristine Ta2O5-x mesocrystals, which could provide more surface reactive sites and high electron density center for trapping photo-generated holes. Besides, possible tetracycline transformation pathways over surface engineered Ta2O5-x mesocrystals and visible light photocatalytic mechanism were also proposed in this work. Current work also provides a facile strategy for regulating surface property of ultrawide bandgaps semiconductors for enhanced visible light photocatalytic performance.


Assuntos
Luz , Tetraciclina , Antibacterianos , Catálise , Propriedades de Superfície
5.
Proc Natl Acad Sci U S A ; 118(15)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876763

RESUMO

Complex II, also known as succinate dehydrogenase (SQR) or fumarate reductase (QFR), is an enzyme involved in both the Krebs cycle and oxidative phosphorylation. Mycobacterial Sdh1 has recently been identified as a new class of respiratory complex II (type F) but with an unknown electron transfer mechanism. Here, using cryoelectron microscopy, we have determined the structure of Mycobacterium smegmatis Sdh1 in the presence and absence of the substrate, ubiquinone-1, at 2.53-Å and 2.88-Å resolution, respectively. Sdh1 comprises three subunits, two that are water soluble, SdhA and SdhB, and one that is membrane spanning, SdhC. Within these subunits we identified a quinone-binding site and a rarely observed Rieske-type [2Fe-2S] cluster, the latter being embedded in the transmembrane region. A mutant, where two His ligands of the Rieske-type [2Fe-2S] were changed to alanine, abolished the quinone reduction activity of the Sdh1. Our structures allow the proposal of an electron transfer pathway that connects the substrate-binding and quinone-binding sites. Given the unique features of Sdh1 and its essential role in Mycobacteria, these structures will facilitate antituberculosis drug discovery efforts that specifically target this complex.

6.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843629

RESUMO

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Succinato Desidrogenase/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Transporte de Elétrons , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
7.
J Colloid Interface Sci ; 572: 141-150, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32240787

RESUMO

Harvesting broad spectral absorption and visible light photocatalysis of ultrawide bandgap semiconductors are one of most significative topics in the solar energy conversion and utilization fields. In this work, amorphous Cl-Ta2O5-x microspheres were prepared by facile solvothermal method for stabilized visible light photocatalytic hydrogen generation. The acetone absorbed on the interfaces of Ta2O5 nanoparticles induced the formation of oxygen vacancies, enhanced visible light absorption, and formation of Ta2O5-x microspheres with preferred orientations as well as Cl doping. The Cl-Ta2O5-x microspheres showed typical amorphous characteristics and obvious visible light absorption in comparison to those of commercial Ta2O5. More importantly, the prepared Cl-Ta2O5-x microspheres also showed stabilized visible light photocatalytic hydrogen generation performance in the spectral regions of 400 nm ≤ λ ≤ 600 nm mainly because of the introduction of oxygen vacancy defects and Cl doping, which might significantly expand the application of tantalum oxide semiconductors in the broad spectral photocatalytic water splitting.

8.
ACS Appl Mater Interfaces ; 12(3): 3919-3927, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31891479

RESUMO

Gas sensors with high sensitivity, fast response/recovery, good selectivity, and room-temperature operation are highly desirable for practical use. However, most of the existing gas sensing materials, either conventional metal oxide semiconductors or advanced inorganic two-dimensional (2D) polymers, can hardly satisfy the above requirements. Herein, we demonstrate an organic 2D polymer derived from a covalent triazine framework (CTF), which possesses nanoscale thickness, intrinsic and periodic pore structures, and abundant functional groups with excellent gas sensing performance. The as-prepared triazine-based 2D polymer (T-2DP) exhibits selective recognition to NO2 with an ultrahigh sensitivity of 452.6 ppm-1, which outperforms most other 2D nanomaterials and its CTF matrix. The sensing effect is superfast (35-47 s) and fully reversible operated at room temperature. The superior comprehensive gas sensing performance of T-2DP and the underlying mechanism was experimentally studied and further discussed by comparison with that of CTF and widely investigated inorganic 2D polymers including graphene and MXene. As a proof of concept, a flexible NO2 chemiresistor based on T-2DP was fabricated to demonstrate its potential for integration into wearable electronics. The scientific findings in this work may propose a new route for the design of high-performance gas sensing materials on the basis of organic 2D polymers in next-generation wearable electronic devices.

9.
J Colloid Interface Sci ; 560: 359-368, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31635883

RESUMO

Highly ordered mesocrystalline semiconductors often indicate tremendous prospects in the clean energy production and environmental photocatalysis mainly because of their unique superstructure for efficient charge transport pathways and long-lived charges. Here, superstructure Ta3N5 mesocrystals with the high-energy surface {2 0 0} planes exposed were the first time to be successfully fabricated by topological transformation of Ta2O5 mesocrystals. The prepared Ta3N5 mesocrystals showed enhanced visible-light photocatalytic hydrogen production activity of 98.67 µmol g-1 for 180 min irradiation, which was approximately 5.28 times that of comm-Ta3N5 prepared with commercial Ta2O5 as the starting material, mainly due to the formation of long-distance electron conduction pathways and long-lived charges. The detailed electronic band structures of the prepared Ta3N5 mesocrystals were also investigated by electrochemical method. Finally, possible visible-light photocatalytic mechanisms of Ta3N5 mesocrystals for enhanced hydrogen production was also proposed in detail. Current work also indicates that tantalum-based mesocrystals show great potential to enhance the charge separation for efficient photocatalytic water splitting.

10.
ACS Appl Mater Interfaces ; 11(20): 18645-18653, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31042350

RESUMO

Highly sensitive mechanical sensing is vital for the emerging field of skin mimicry and wearable healthcare systems. To date, it remains a big challenge to fabricate mechanosensors with both high sensitivity and a wide sensing range. In nature, slit sensilla are crack-shaped sensory organs of arachnids, which are highly sensitive to tiny external mechanical stimuli. Here, inspired by the geometry of slit sensilla, a concept is developed that pretextures reduced graphene oxide (RGO) nanocoating into multiscale topographies with agminated crumples and interlaced cracks (crumpled & cracked RGO) through an efficient and scalable mechanically driven process. Both the sensitivity and the workable range can be facilely tuned by adjusting the crack density. The resulting mechanosensor exhibits a comprehensive superior performance including high sensitivity (a gauge factor of 205 to 3256), a wide and tunable sensing range (from 0-40 to 0-180%), long-term stability (over 5000 cycles), and multiple sensing functions. Based on its excellent performances, the mechanosensor can be used as a wearable electronic to in situ monitor subtle physiological signals and vigorous body actions. The rationally designed crumpled & cracked RGO provides a promising platform for artificial electronic skin and portable healthcare systems.


Assuntos
Grafite , Dispositivos Eletrônicos Vestíveis , Propriedades de Superfície
11.
Science ; 362(6418)2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30361386

RESUMO

We report a 3.5-angstrom-resolution cryo-electron microscopy structure of a respiratory supercomplex isolated from Mycobacterium smegmatis. It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis.


Assuntos
Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Transporte de Elétrons , Mycobacterium smegmatis/enzimologia , Superóxido Dismutase/química , Actinobacteria/enzimologia , Microscopia Crioeletrônica , Oxirredução , Fosforilação Oxidativa , Oxigênio/metabolismo , Multimerização Proteica
12.
J Mol Histol ; 48(5-6): 427-436, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29094227

RESUMO

Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57 ± 10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.


Assuntos
Proteínas de Transporte/metabolismo , Regulação para Baixo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células , Regulação para Baixo/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , Camundongos , MicroRNAs/genética , Oligonucleotídeos/metabolismo , Osteogênese/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo
13.
Chem Commun (Camb) ; 50(64): 8834-7, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24840743

RESUMO

The preferentially (h0l)-oriented beta zeolite membrane was prepared on the porous α-Al2O3 support by secondary growth of a beta seed layer in the absence of organic templates.

14.
Anal Chem ; 85(16): 7957-65, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23863032

RESUMO

Mass spectrometry-based platforms have gained increasing success in discovery of ligands bound to therapeutic targets as drug candidates. We established both a nanoelectrospray ionization mass spectrometry (nanoESI-MS) assay and an ultrafiltration liquid chromatography/mass spectrometry (LC/MS) assay to identify new ligands for New Delhi metallo-ß-lactamase 1 (NDM-1), responsible for worldwide antibiotic resistance. To alleviate nonspecific binding of hydrophobic compounds and eliminate false positives typically encountered in the indirect LC/MS-based assay, we introduced a blocking protein in the control, which remarkably enhances the selectivity and accuracy of the indirect approach. Side-by-side comparison of the two MS-based approaches for the first time further reveals unique advantages of the indirect approach, including better reproducibility and tolerance of interference. Moreover, the success of fishing out a potent ligand from a mixture of small-molecule fragments demonstrates great potential of the indirect LC/MS-based approach for constructing a robust screening platform against combinatorial libraries or natural product extracts. More importantly, by combining the results of MS-based analyses, enzymatic activity assay, competition experiments, and structural simulation, we discovered a new compound as a promising drug candidate targeting NDM-1.


Assuntos
Antibacterianos/farmacologia , Cromatografia Líquida/métodos , Resistência Microbiana a Medicamentos , Inibidores Enzimáticos/farmacologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Ultrafiltração/métodos , beta-Lactamases/análise , Nanotecnologia
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