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1.
Plant Physiol Biochem ; 141: 325-331, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207493

RESUMO

Type 2C protein phosphatases (PP2Cs) counteract protein kinases, thereby inhibiting the abscisic acid (ABA)-mediated response to abiotic stress in Arabidopsis thaliana. In the absence of stress, the promoters of PP2C genes (e.g., ABI1, ABI2, and HAI1) are negatively regulated by repressors that suppress gene transcription in a signal-independent manner. Quantitative reverse transcription PCR (RT-qPCR) and chromatin immunoprecipitation (ChIP) assays revealed that the levels of PP2C gene transcripts and RNA polymerase II (RNAPII) that stalled at the transcription start sites (TSS) of PP2C gene loci were increased under salt stress. The salt-induced increases in RNA polymerase-mediated transcription were reduced in 35S:AtMYB44 plants, confirming that AtMYB44 acts as a repressor of PP2C gene transcription. ChIP assays revealed that AtMYB44 repressors are released and nucleosomes are evicted from the promoter regions in response to salt stress. Under these conditions, histone H3 acetylation (H3ac) and methylation (H3K4me3) around the TSS regions significantly increased. The salt-induced increases in PP2C gene transcription were reduced in abf3 plants, indicating that ABF3 activates PP2C gene transcription. Overall, our data indicate that salt stress converts PP2C gene chromatin from a repressor-associated suppression status to an activator-mediated transcription status. In addition, we observed that the Arabidopsis mutant brm-3, which is moderately defective in SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) activity, produced more PP2C gene transcripts under salt stress conditions, indicating that BRM ATPase contributes to the repression of PP2C gene transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/química , Nucleossomos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Estresse Salino , Trifosfato de Adenosina/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
2.
J Chem Theory Comput ; 15(5): 3362-3380, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31017783

RESUMO

In this work, we have examined the molecular mechanisms of allosteric regulation of the ABL tyrosine kinase at the atomic level. Atomistic modeling of the ABL complexes with a panel of allosteric modulators has been performed using a combination of molecular dynamics simulations, structural residue perturbation scanning, and a novel community analysis of the residue interaction networks. Our results have indicated that allosteric inhibitors and activators may exert a differential control on allosteric signaling between the kinase binding sites and functional regions. While the inhibitor binding can strengthen the closed ABL state and induce allosteric communications directed from the allosteric pocket to the ATP binding site, the DPH activator may induce a more dynamic open form and activate allosteric couplings between the ATP and substrate binding sites. By leveraging a network-centric theoretical framework, we have introduced a novel community analysis method and global topological parameters that have unveiled the hierarchical modularity and the intercommunity bridging sites in the residue interaction network. We have found that allosteric functional hotspots responsible for the kinase regulation may serve the intermodular bridges in the global interaction network. The central conclusion from this analysis is that the regulatory switch centers play a fundamental role in the modular network organization of ABL as the unique intercommunity bridges that connect the SH2 and SH3 domains with the catalytic core into a functional kinase assembly. The hierarchy of network organization in the ABL regulatory complexes may allow for the synergistic action of dense intercommunity links required for the robust signal transfer in the catalytic core and sparse network bridges acting as the regulatory control points that orchestrate allosteric transitions between the inhibited and active kinase forms.


Assuntos
Simulação de Dinâmica Molecular , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-abl/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Humanos , Conformação Proteica , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química
3.
Nat Commun ; 10(1): 1720, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979890

RESUMO

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.


Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/análise , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Animais , Tampões (Química) , DNA Helicases/química , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Humanos , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Xenopus
4.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31032817

RESUMO

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.


Assuntos
Enzimas/química , Enzimas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Celulase/química , Celulase/metabolismo , Fenômenos Químicos , Sequência Conservada , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
5.
Nat Commun ; 10(1): 1155, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30858367

RESUMO

Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteoma/metabolismo , Trifosfato de Adenosina/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Células Jurkat , Espectrometria de Massas , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteoma/química , Proteômica/métodos , Solubilidade
6.
Biosensors (Basel) ; 9(1)2019 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-30832416

RESUMO

Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation.


Assuntos
Técnicas Biossensoriais/métodos , Óptica e Fotônica/métodos , Análise Espectral Raman/métodos , Adenosina/química , Adenosina/isolamento & purificação , Trifosfato de Adenosina/química , Trifosfato de Adenosina/isolamento & purificação , Humanos , Interleucina-6/química , Interleucina-6/isolamento & purificação , Dispositivos Lab-On-A-Chip , Limite de Detecção , Fenilalanina/química , Fenilalanina/isolamento & purificação , Lactato de Sódio/química , Lactato de Sódio/isolamento & purificação , Ressonância de Plasmônio de Superfície , Propriedades de Superfície
7.
Molecules ; 24(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832206

RESUMO

In this study, we report on the modification of a 3,4-diaryl-isoxazole-based CK1 inhibitor with chiral pyrrolidine scaffolds to develop potent and selective CK1 inhibitors. The pharmacophore of the lead structure was extended towards the ribose pocket of the adenosine triphosphate (ATP) binding site driven by structure-based drug design. For an upscale compatible multigram synthesis of the functionalized pyrrolidine scaffolds, we used a chiral pool synthetic route starting from methionine. Biological evaluation of key compounds in kinase and cellular assays revealed significant effects of the scaffolds towards activity and selectivity, however, the absolute configuration of the chiral moieties only exhibited a limited effect on inhibitory activity. X-ray crystallographic analysis of ligand-CK1δ complexes confirmed the expected binding mode of the 3,4-diaryl-isoxazole inhibitors. Surprisingly, the original compounds underwent spontaneous Pictet-Spengler cyclization with traces of formaldehyde during the co-crystallization process to form highly potent new ligands. Our data suggests chiral "ribose-like" pyrrolidine scaffolds have interesting potential for modifications of pharmacologically active compounds.


Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/química , Isoxazóis/química , Trifosfato de Adenosina/química , Sítios de Ligação , Caseína Quinase Idelta/química , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Isoxazóis/síntese química , Isoxazóis/farmacologia , Ligantes , Complexos Multiproteicos/química , Pirrolidinas/química , Relação Estrutura-Atividade
8.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836629

RESUMO

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.


Assuntos
Apoenzimas/genética , Simulação de Dinâmica Molecular , Fósforo-Oxigênio Liases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Apoenzimas/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Di-Hidroxiacetona/química , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/química , Gliceraldeído/química , Humanos , Fósforo-Oxigênio Liases/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por Substrato
10.
Nat Commun ; 10(1): 1273, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894538

RESUMO

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.


Assuntos
Adenosina Trifosfatases/química , Chaperoninas/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Chaperoninas/genética , Chaperoninas/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
11.
Talanta ; 198: 1-7, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876536

RESUMO

In this paper, a split aptamer based fluorescence resonance energy transfer (FRET) platform was constructed for the determination of adenosine deaminase (ADA) activity by using gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs). A single adenosine triphosphate (ATP) aptamer was split into two fragments (referred to as P1 and P2). P1 was covalently attached to the AuNCs at the 5' end (P1-AuNCs), and P2 was labeled with AuNPs at the 3' end (P2-AuNPs). In the presence of ATP, ATP bound with the two fragments with high affinity to link P1-AuNCs and P2-AuNPs together, thus the fluorescence of P1-AuNCs was quenched via FRET from P1-AuNCs to P2-AuNPs. With the addition of ADA, ATP was transformed into inosine triphosphate (ITP), and then P1 and P2 were released to cause the fluorescence recovery of the system. So a split aptamer based FRET platform for ADA detection can be established via the fluorescence intensity change of the system. This platform showed a good linear relationship between the fluorescence intensity and ADA concentration in the range of 2-120 U L-1, and the limit of detection (LOD) was 0.72 U L-1. Moreover, the detection of ATP in human serum sample demonstrated the accuracy and applicability of the method for ADA detection in real sample.


Assuntos
Adenosina Desaminase/sangue , Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Transferência Ressonante de Energia de Fluorescência , Adenosina Desaminase/metabolismo , Ouro/química , Humanos , Nanopartículas Metálicas/química , Tamanho da Partícula , Propriedades de Superfície
12.
Environ Sci Pollut Res Int ; 26(13): 12842-12850, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30888618

RESUMO

The emission of nitrogen oxides has caused severe harm to the ecosystem; thus, the development of low-cost and high-efficiency denitrification catalysts and new methods are of great significance. In this work, a co-precipitation method was employed to prepare Pr-doped CeO2/attapulgite (CeO2/Pr3+/ATP) nanocomposites. X-ray diffraction (XRD), photoluminance spectroscopy (PL), ultraviolet-visible diffuse reflectance (UV-Vis), Fourier transform infrared (FT-IR), and high-resolution transmission electron microscopy (HRTEM) were utilized to characterize the products. Results showed that the CeO2/Pr3+ nanoparticles were uniformly coated on the surface of ATP and demonstrated outstanding upconversion effect which converted the visible light to ultraviolet light. The upconversion luminescence of CeO2/Pr3+/ATP was strongest when the molar doping amount of Pr was 1 mol%, and the photo-SCR denitrification achieved the highest of 90% conversion and 95% selectivity when the loading amount of CeO2/Pr3+ was 40 wt%. The ATP and CeO2/Pr3+ constructed an indirect Z-type heterojunction structure mediated by oxygen vacancy which benefited the separation of charge carriers and enhanced the reduction-oxidation potentials, both are responsible for the remarkable denitrification performance.


Assuntos
Cério/química , Compostos de Magnésio/química , Nanocompostos/química , Óxidos de Nitrogênio/isolamento & purificação , Praseodímio/química , Compostos de Silício/química , Trifosfato de Adenosina/química , Catálise , Precipitação Química , Desnitrificação , Luz , Luminescência , Óxidos de Nitrogênio/química , Oxigênio/química , Fotoquímica/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Raios Ultravioleta , Difração de Raios X
13.
Biosens Bioelectron ; 133: 32-38, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30904620

RESUMO

Inspired by the design of folding greeting cards and tissue drawing covers, a photoelectrochemical (PEC) lab-on-paper device with a controllable fluid separator, producing both reaction zone and detection zone, was explored for ultrasensitive detection of adenosine 5'-triphosphate (ATP) via mimic peroxidase-transfer enhancement of photocurrent response. To realize it, the DNA1, aptamer, and DNA2 as well as the mimic peroxidase of G-quadruplex/hemin modified Au nanocubes were linked on the graphene oxide-functionalized reaction zone via the DNA hybridization. Meanwhile, three-dimensional CuO nanoflowers (CuO NFs) as a photoactive material with outstanding electron transfer ability and absorption of light were grown in situ on the detection zone, providing a PEC active interface. Besides, an innovative fluid separator was elaborately designed by assembling a strip of paper with a hydrophilic channel, providing an effective way to bridge the gap between the two zones with a controllable drawing way, which could successfully avoid the signal interference caused by modifying biomolecules layer by layer on photosensitive materials. In the presence of ATP, the G-quadruplex/hemin modified in the reaction zone was dissociated due to the specific recognition of ATP with aptamer and released into the detection zone with the assistance of controllable fluid separator. The free G-quadruplex/hemin could catalyze hydrogen peroxide to generate oxygen for the consumption of photo-induced electrons from CuO NFs, which could further promote the electron-hole carriers separation efficiency, and eventually resulting in the enhancement of PEC signal. The proposed PEC lab-on-paper device could be employed for specific detection of ATP in the range from 5.0 to 3.0 × 103 nM with a detection limit of 2.1 nM.


Assuntos
Trifosfato de Adenosina/isolamento & purificação , Técnicas Biossensoriais , Técnicas Eletroquímicas , Trifosfato de Adenosina/química , Quadruplex G , Grafite/química , Hemina/química , Peróxido de Hidrogênio/química , Limite de Detecção , Nanoestruturas/química , Hibridização de Ácido Nucleico , Peroxidases/química , Processos Fotoquímicos , Pontos Quânticos/química
14.
J Zhejiang Univ Sci B ; 20(3): 219-237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30829010

RESUMO

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in cancer development and progression. However, the mechanism by which they enhance the chemoresistance of ovarian cancer is unknown. METHODS: Conditioned media of BM-MSCs (BM-MSC-CM) were analyzed using a technique based on microRNA arrays. The most highly expressed microRNAs were selected for testing their effects on glycolysis and chemoresistance in SKOV3 and COC1 ovarian cancer cells. The targeted gene and related signaling pathway were investigated using in silico analysis and in vitro cancer cell models. Kaplan-Merier survival analysis was performed on a population of 59 patients enrolled to analyze the clinical significance of microRNA findings in the prognosis of ovarian cancer. RESULTS: MiR-1180 was the most abundant microRNA detected in BM-MSC-CM, which simultaneously induces glycolysis and chemoresistance (against cisplatin) in ovarian cancer cells. The secreted frizzled-related protein 1 (SFRP1) gene was identified as a major target of miR-1180. The overexpression of miR-1180 led to the activation of Wnt signaling and its downstream components, namely Wnt5a, ß-catenin, c-Myc, and CyclinD1, which are responsible for glycolysis-induced chemoresistance. The miR-1180 level was inversely correlated with SFRP1 mRNA expression in ovarian cancer tissue. The overexpressed miR-1180 was associated with a poor prognosis for the long-term (96-month) survival of ovarian cancer patients. CONCLUSIONS: BM-MSCs enhance the chemoresistance of ovarian cancer by releasing miR-1180. The released miR-1180 activates the Wnt signaling pathway in cancer cells by targeting SFRP1. The enhanced Wnt signaling upregulates the glycolytic level (i.e. Warburg effect), which reinforces the chemoresistance property of ovarian cancer cells.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Via de Sinalização Wnt , Trifosfato de Adenosina/química , Adulto , Idoso , Células da Medula Óssea/citologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Feminino , Citometria de Fluxo , Seguimentos , Glicólise , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Análise Multivariada , Regulação para Cima
15.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
16.
Anticancer Res ; 39(2): 695-701, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30711947

RESUMO

BACKGROUND/AIM: Sorafenib is now standard treatment for advanced hepatocellular carcinoma (HCC). However, therapeutic efficacy is not as good as was predicted. Many efforts are being made to improve HCC sensitivity to sorafenib. Our previous study demonstrated that co-treatment with chrysin enhanced sorafenib sensitivity through inhibition of ATP-binding cassette super-family G member 2 (ABCG2). Whether there is another mechanism other than inhibition of ABCG2 underlying chrysin-mediated synergistic effect is still not completely elucidated. MATERIALS AND METHODS: Phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was examined by western blot. Cell viability was examined by crystal violet staining. The importance of ERK1/2 phosphorylation was assessed by overexpression and blockage of mitogen-activated protein kinase kinase 1 (MEK1). RESULTS: Chrysin induced sustained ERK1/2 phosphorylation of HCC cells in both time- and dose-dependent manners. Overexpression of MEK1 enhanced, whereas blockage of MEK1 led to loss of chrysin-synergized sorafenib effect, through modulating ERK1/2 phosphorylation level. CONCLUSION: These results identify another novel mechanism underlying chrysin-mediated synergistic effect on sorafenib activity in HCC cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Hepatocelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Neoplasias Hepáticas/patologia , Sorafenibe/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/química , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação
17.
Org Biomol Chem ; 17(9): 2423-2431, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30735219

RESUMO

Mevalonate Kinase (MVK) catalyses the ATP-Mg2+ mediated phosphate transfer of mevalonate to produce mevalonate 5-phosphate and is a key kinase in the mevalonate pathway in the biosynthesis of isopentenyl diphosphate, the precursor of isoprenoid-based biofuels. However, the crystal structure in complex with the native substrate mevalonate, ATP and Mg2+ has not been resolved, which has limited the understanding of its reaction mechanism and therefore its application in the production of isoprenoid-based biofuels. Here using molecular docking, molecular dynamics (MD) simulations and a hybrid QM/MM study, we revisited the location of Mg2+ resolved in the crystal structure of MVK and determined a catalytically competent MVK structure in complex with the native substrate mevalonate and ATP. We demonstrated that significant conformational change on a flexible loop connecting the α6 and α7 helix is induced by the substrate binding. Further, we found that Asp204 is coordinated to the Mg2+ ion. Arg241 plays a crucial role in organizing the triphosphoryl tail of ATP for in-line phosphate transfer and stabilizing the negative charge that accumulates at the ß,γ-bridging oxygen of ATP upon bond cleavage. Remarkably, we revealed that the phosphorylation of mevalonate catalyzed by MVK occurs via a direct phosphorylation mechanism, instead of the conventionally postulated catalytic base mechanism. The catalytically competent complex structure of MVK as well as the mechanism of reaction will pave the way for the rational engineering of MVK to exploit its applications in the production of biofuels.


Assuntos
Ácido Mevalônico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Magnésio/química , Magnésio/metabolismo , Ácido Mevalônico/química , Simulação de Acoplamento Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Teoria Quântica , Ratos
18.
Chemistry ; 25(20): 5158-5162, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30791173

RESUMO

Biological macromolecular machines perform impressive mechanical movements. F-adenosine triphosphate (ATP) synthase uses a proton gradient to generate ATP through mechanical rotations. Here, a programmed hexagonal DNA nanomachine, in which a three-armed DNA nanostructure (TAN) can perform stepwise rotations in the confined nanospace powered by DNA fuels, is demonstrated. The movement of TAN can precisely go through a 60° rotation, which is confirmed by atomic force microscopy, and each stepwise directional rotating is monitored by fluorescent measurements. Moreover, the rotary nanomachine is used to spatially organize cascade enzymes: glucose oxidase (GOx) and horseradish peroxidase (HRP) in four different arrangements. The multistep regulations of the biocatalytic activities are achieved by employing TAN rotations. This work presents a new prototype of rotary nanodevice with both angular and directional control, and provides a nanoscale mechanical engineering platform for the reactive molecular components, demonstrating that DNA-based framework may have significant roles in futuristic nanofactory construction.


Assuntos
DNA/química , Nanoestruturas/química , Trifosfato de Adenosina/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Nanotecnologia , Rotação
19.
Nat Commun ; 10(1): 711, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755613

RESUMO

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Citosol/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/metabolismo , Trifosfato de Adenosina/genética , Bacillus/citologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Cinética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
20.
Molecules ; 24(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704145

RESUMO

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αß)3δab2. Subunits c10 or (αß)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αß)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme's rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ouro , Cinética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Nanotubos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
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