Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.478
Filtrar
1.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
2.
Nat Commun ; 11(1): 2478, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424158

RESUMO

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 Å resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Esterol O-Aciltransferase/química , Sítios de Ligação , Biocatálise , Células HEK293 , Humanos , Ligantes , Multimerização Proteica , Esterol O-Aciltransferase/ultraestrutura , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos
3.
Biochem J ; 477(2): 431-444, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904830

RESUMO

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.


Assuntos
Aurora Quinase A/genética , Caseína Quinase II/química , Fosfoproteínas Fosfatases/genética , Alanina/genética , Substituição de Aminoácidos/genética , Aurora Quinase A/química , Sistemas CRISPR-Cas/genética , Caseína Quinase II/genética , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Especificidade por Substrato/efeitos dos fármacos
4.
J Biol Chem ; 295(8): 2464-2472, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31953328

RESUMO

Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (Ki < 2 nm). Using bio-layer interferometry-based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site-directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.


Assuntos
Metaloproteinase 10 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Camelídeos Americanos , Humanos , Imunização , Cinética , Biblioteca de Peptídeos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/efeitos dos fármacos , alfa 1-Antitripsina/metabolismo
5.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936522

RESUMO

ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.


Assuntos
Alteromonadaceae/enzimologia , Organismos Aquáticos/enzimologia , beta-N-Acetil-Hexosaminidases/biossíntese , Alteromonadaceae/genética , Organismos Aquáticos/genética , Biocatálise/efeitos dos fármacos , Estabilidade Enzimática , Genoma Bacteriano , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Octoxinol/farmacologia , Filogenia , Domínios Proteicos , Soroalbumina Bovina/farmacologia , Cloreto de Sódio/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Fatores de Tempo , beta-N-Acetil-Hexosaminidases/química
6.
Nat Commun ; 10(1): 5566, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804482

RESUMO

Overexpressed Aurora-A kinase promotes tumor growth through various pathways, but whether Aurora-A is also involved in metabolic reprogramming-mediated cancer progression remains unknown. Here, we report that Aurora-A directly interacts with and phosphorylates lactate dehydrogenase B (LDHB), a subunit of the tetrameric enzyme LDH that catalyzes the interconversion between pyruvate and lactate. Aurora-A-mediated phosphorylation of LDHB serine 162 significantly increases its activity in reducing pyruvate to lactate, which efficiently promotes NAD+ regeneration, glycolytic flux, lactate production and bio-synthesis with glycolytic intermediates. Mechanistically, LDHB serine 162 phosphorylation relieves its substrate inhibition effect by pyruvate, resulting in remarkable elevation in the conversions of pyruvate and NADH to lactate and NAD+. Blocking S162 phosphorylation by expression of a LDHB-S162A mutant inhibited glycolysis and tumor growth in cancer cells and xenograft models. This study uncovers a function of Aurora-A in glycolytic modulation and a mechanism through which LDHB directly contributes to the Warburg effect.


Assuntos
Aurora Quinase A/metabolismo , Glicólise , L-Lactato Desidrogenase/metabolismo , Animais , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Pirimidinas/farmacologia , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Serina/genética , Serina/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Molecules ; 24(17)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31480528

RESUMO

Cytochromes P450 are major metabolic enzymes involved in the biotransformation of xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are exposed through everyday diet. In the previous study, selected flavonoid aglycones showed inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose, the O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated metabolites were monitored by high-performance liquid chromatography coupled with diode array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/farmacologia , Sistema Enzimático do Citocromo P-450/química , Flavonoides/química , Humanos , Simulação de Acoplamento Molecular , Especificidade por Substrato/efeitos dos fármacos
8.
Mol Pharmacol ; 96(5): 629-640, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31515284

RESUMO

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Citotoxinas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia
9.
J Pharmacol Exp Ther ; 371(2): 320-326, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31455631

RESUMO

CC-115, a triazole-containing compound, is a dual mammalian target of rapamycin (mTOR)/DNA-dependent protein kinase (DNA-PK) inhibitor currently in clinical trials. To develop this compound further, we investigated factors that may affect cellular response to CC-115. Previously, fatty acid synthase (FASN) was shown to upregulate DNA-PK activity and contribute to drug resistance; therefore, we hypothesized that FASN may affect cellular response to CC-115. Instead, however, we showed that CC-115 is a substrate of ATP-binding cassette G2 (ABCG2), a member of the ATP-binding cassette transporter superfamily, and that expression of ABCG2, not FASN, affects the potency of CC-115. ABCG2 overexpression significantly increases resistance to CC-115. Inhibiting ABCG2 function, using small-molecule inhibitors, sensitizes cancer cells to CC-115. We also found that CC-115 may be a substrate of ABCB1, another known ABC protein that contributes to drug resistance. These findings suggest that expression of ABC transporters, including ABCB1 and ABCG2, may affect the outcome in clinical trials testing CC-115. Additionally, the data indicate that ABC transporters may be used as markers for future precision use of CC-115. SIGNIFICANCE STATEMENT: In this article, we report our findings on the potential mechanism of resistance to CC-115, a dual inhibitor of mTOR and DNA-PK currently in clinical trials. We show that CC-115 is a substrate of ABCG2 and can be recognized by ABCB1, which contributes to CC-115 resistance. These findings provide novel information and potential guidance on future clinical testing of CC-115.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Triazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ensaios Clínicos como Assunto/métodos , DNA/antagonistas & inibidores , DNA/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos/fisiologia , Células HEK293 , Humanos , Células MCF-7 , Fatores de Risco , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia
10.
Int J Biol Macromol ; 140: 949-958, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445147

RESUMO

In this study, hydrazine treated acrylic fabrics (polyacrylonitrile, PAN) activated with cyanuric chloride was developed as supporting material for horseradish peroxidase (HRP) immobilization. The immobilization of HRP onto the modified supporting material was achieved after being end-over-end incubated for 12 h. Field emission scanning electron microscopy and Fourier-transform infrared spectroscopy techniques were used to confirm the successful immobilization. Reusability experiment was performed to estimate the ability of the immobilized HRP to recover the reaction medium, in which it was observed to retain 78% of its original activity after 10 cycles. Relative to the soluble HRP, the optimum pH and temperature for the immobilized HRP were shifted to 7-7.5 and 50 °C, respectively. The kinetic parameters of guaiacol and H2O2 for the immobilized HRP were determined to be Km/Vmax = 57.61, 11.35 and Kcat/Km = 1.87, 1.86, respectively, while the values for the free form were Km/Vmax = 41.49, 6.23 and Kcat/Km = 1.87, 1.86, respectively. Compared to the soluble form, the immobilized HRP exhibited higher resistance toward metal ions and some organic solvents. For an application perspective. The immobilization of HRP using this procedure has the potential to be used for industrial application and wastewater treatment.


Assuntos
Resinas Acrílicas/química , Enzimas Imobilizadas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Fenol/isolamento & purificação , Triazinas/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Hidrazinas/química , Hidrazinas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Fatores de Tempo , Triazinas/química
11.
Proc Natl Acad Sci U S A ; 116(32): 15895-15900, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337679

RESUMO

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.


Assuntos
Produtos Biológicos/química , Calmodulina/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Cálcio/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Quinase 5 de Receptor Acoplado a Proteína G/química , Hipertrofia , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos
12.
Aquat Toxicol ; 214: 105238, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301544

RESUMO

Abiotic stresses enhance the cellular level of reactive oxygen species (ROS) which consequently leads to toxic methylglyoxal (MG) production. Glyoxalases (GlyI & GlyII) catalyze the conversion of toxic MG into non-toxic lactic acid but their properties and functions have been overlooked in cyanobacteria. This is the first attempt to conduct a genome-wide analysis of GlyI protein (PF00903) from Anabaena sp. PCC7120. Out of total nine GlyI domain possessing proteins, only three (Alr2321, Alr4469, All1022) harbour conserve His/Glu/His/Glu metal binding site at their homologous position and are deficient in conserved region specific for Zn2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni2+ dependent active GlyI with catalytic efficiency 11.7 × 106 M-1 s-1. It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni2+ followed by Co2+ > Mn2+ > Cu2+ and no activity with Zn2+. Moreover, the expression of alr2321 was found to be maximally up-regulated under heat (19 fold) followed by cadmium, desiccation, arsenic, salinity and UV-B stresses. BL21/pGEX-5X2-alr2321 showed improved growth under various abiotic stresses as compared to BL21/pGEX-5X2 by increased scavenging of intracellular MG and ROS levels. Taken together, these results suggest noteworthy links between intracellular MG and ROS, its detoxification by Alr2321, a member of GlyI family of Anabaena sp. PCC7120, in relation to abiotic stress.


Assuntos
Anabaena/enzimologia , Lactoilglutationa Liase/metabolismo , Aldeído Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Anabaena/efeitos dos fármacos , Inativação Metabólica/efeitos dos fármacos , Cinética , Lactoilglutationa Liase/química , Lactoilglutationa Liase/genética , Metais/farmacologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato/efeitos dos fármacos
13.
Biomed Pharmacother ; 117: 109059, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207578

RESUMO

Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance.


Assuntos
Doxorrubicina/farmacologia , Teratogênios/toxicidade , Alcaloides de Veratrum/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Células MCF-7 , Estrutura Secundária de Proteína , Especificidade por Substrato/efeitos dos fármacos , Teratogênios/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia
14.
Methods Mol Biol ; 1988: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147928

RESUMO

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.


Assuntos
Bioensaio/métodos , Citosol/enzimologia , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Centrifugação com Gradiente de Concentração , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Eritrócitos/enzimologia , Corantes Fluorescentes/metabolismo , Humanos , Inibidores de Proteassoma/farmacologia , Especificidade por Substrato/efeitos dos fármacos
15.
Int Wound J ; 16(4): 1013-1023, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31148413

RESUMO

Bacterial collagenase from the aerobic non-pathogenic Vibrio alginolyticus chemovar iophagus is an extracellular metalloproteinase. This collagenase preparation is obtained through a fermentation process and is purified chromatographically, resulting in a highly purified 82-kDa single-band protein that does not contain non-specific proteases or other microbial impurities. V. alginolyticus collagenase was added to a hyaluronan (HA)-based device to develop a novel debriding agent to improve the treatment of ulcers, necrotic burns, and decubitus in the initial phase of wound bed preparation. In this study, an in vitro biochemical characterisation of V. alginolyticus collagenase versus a commercial preparation from a Clostridium histolyticum strain on various dermal extracellular matrix (ECM) substrates was performed. V. alginolyticus collagenase demonstrated its ability to carry out the enzymatic cleavage of the substrate, allowing a selective removal of necrotic tissues while sparing healthy tissue, as reported in clinical studies and through routine clinical experience. in vitro tests under physiological conditions (pH, presence of Ca++, etc.) have demonstrated that V. alginolyticus collagenase exhibits very poor/limited non-specific proteolytic activity, whereas the collagenase preparation from C. histolyticum is highly active both on collagen and on non-collagenic substrates. This finding implies that while the V. alginolyticus enzyme is fully active on the collagen filaments that anchor the necrotic tissue to the wound bed, it does not degrade other minor, but structurally important, components of the dermal ECM. This feature could explain why collagenase preparation from V. alginolyticus has been reported to be much gentler on perilesional, healthy skin.


Assuntos
Colagenases/química , Colagenases/uso terapêutico , Colagenase Microbiana/química , Colagenase Microbiana/uso terapêutico , Especificidade por Substrato/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Clostridium histolyticum/química , Humanos , Vibrio alginolyticus/química
16.
New Phytol ; 223(4): 1904-1917, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31087404

RESUMO

Choline kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/ETHANOLAMINE KINASE 1 (CEK1) encodes functional CEK that prefers choline than ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/enzimologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Colina/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Marcadores Genéticos , Análise do Fluxo Metabólico , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Plântula/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
17.
PLoS One ; 14(4): e0216077, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31039204

RESUMO

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.


Assuntos
Basidiomycota/enzimologia , Colinesterases/metabolismo , Animais , Basidiomycota/genética , Inibidores da Colinesterase/farmacologia , Colinesterases/genética , Drosophila melanogaster/enzimologia , Genes Fúngicos , Lipase/metabolismo , Filogenia , Especificidade por Substrato/efeitos dos fármacos
18.
Biotechnol Appl Biochem ; 66(4): 607-616, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31056790

RESUMO

Lipases are surface-active enzymes, acting on their substrates at the polar/nonpolar interface in emulsions. This study was aimed to test whether their activity, specificity, and the rates of formation/degradation of the various hydrolysis intermediates (i.e., mono- and diglycerides of interest as surface-active agents) could be modulated by adhesion of the triglyceride substrates as a thin layer on the surface of solids. These hypotheses were tested by using an array of food-grade lipases used in bakery, testing various types of starch as the "solid" phase. Starch-dependent increase in the hydrolysis rate was tested by pH-stat techniques on pure triglycerides and on food-grade oils in diluted emulsions. Starch-related improvements in the rate of fatty acids release were most evident at temperatures above 40 °C, and when using maize starch instead of wheat starch. Starch-dependent changes in the nature of the hydrolysis products were tested by chromatographic profiling of ethyl ether extracts from aqueous slurries containing up to 33% fat and 33% starch. Accumulation of mono- and diglycerides as hydrolysis intermediates was found to be modulated by the type of oil being used, by the reaction conditions, as well as by the enzyme nature and amount.


Assuntos
Lipase/metabolismo , Amido/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Hidrólise/efeitos dos fármacos , Cinética , Lipase/química , Amido/química , Amido/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Triglicerídeos/química , Triglicerídeos/metabolismo
19.
Mol Cell Proteomics ; 18(6): 1197-1209, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926672

RESUMO

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Oxigênio/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Genética/efeitos dos fármacos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Ligação Proteica/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Fator de Transcrição AP-2/química , Fator de Transcrição AP-2/metabolismo
20.
Nat Commun ; 10(1): 1402, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926793

RESUMO

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA