Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 123
Filtrar
1.
Biochem Biophys Res Commun ; 587: 24-28, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34864391

RESUMO

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.


Assuntos
Aliivibrio fischeri/enzimologia , Proteínas de Bactérias/metabolismo , Imidazóis/metabolismo , Luciferases/metabolismo , NADH NADPH Oxirredutases/metabolismo , Pirazinas/metabolismo , Renilla/enzimologia , Aliivibrio fischeri/genética , Animais , Proteínas de Bactérias/genética , Biocatálise , Biotransformação , Cromatografia Líquida de Alta Pressão , Mononucleotídeo de Flavina/metabolismo , Expressão Gênica , Cinética , Luciferases/genética , Luminescência , Medições Luminescentes , NADH NADPH Oxirredutases/genética , Ácidos Fenilpirúvicos/metabolismo , Renilla/genética
2.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374392

RESUMO

Two G-quadruplex forming oligonucleotides [d(TG4T)4 and d(TG6T)4] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3'- and 5'-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines.


Assuntos
Quadruplex G , Lipídeos/química , Nanoestruturas/química , Oligonucleotídeos/genética , Animais , Biofísica , Linhagem Celular Tumoral , Dicroísmo Circular , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos Antissenso , Renilla/enzimologia , Transfecção
3.
J Photochem Photobiol B ; 210: 111980, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32745950

RESUMO

The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1-3 enzymes confer oxygen sensitivity to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of the PHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic inflammation. Several pharmacologic PHD inhibitors (PHIs) are available, but our understanding of their selectivity and its chemical basis is limited. We here report that the PHI JNJ-42041935 (JNJ-1935) is structurally similar to the firefly luciferase substrate D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592 (roxadustat) and FG-2216 (ICA, BIQ, IOX3, YM 311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36 µM. D-luciferin did not inhibit the PHDs, despite its structural similarity to JNJ-1935. This study provides insights into a previously unknown JNJ-1935 off-target effect as well as into the chemical requirements for firefly luciferase and PHD inhibitors and may inform the development of novel compounds targeting these enzymes.


Assuntos
Luciferases de Vaga-Lume/metabolismo , Inibidores de Prolil-Hidrolase/química , Animais , Benzotiazóis/metabolismo , Ligação Competitiva , Vaga-Lumes/enzimologia , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Isoquinolinas/química , Isoquinolinas/metabolismo , Cinética , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Inibidores de Prolil-Hidrolase/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Renilla/enzimologia
4.
Toxicology ; 439: 152476, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32335162

RESUMO

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.


Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Dermatite Alérgica de Contato/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Alternativas aos Testes com Animais , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Ensaio Local de Linfonodo , Luciferases/metabolismo , Renilla/enzimologia , Sensibilidade e Especificidade , Testes Cutâneos , Timidina Quinase/metabolismo
5.
Pharm Dev Technol ; 25(7): 855-864, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32188321

RESUMO

G protein-coupled receptor (GPCR) 87, is overexpressed in various cancer cells especially pancreatic cancer and plays a critical role in tumor cell survival. Nano-particles (NP) have become the essential vehicles for nucleotide internalization to the cell, due to the negative charge of nucleotides and their poor stability in blood circulation. In this study, the HEK293T cell linewas transfected with GPR87-plasmid after which the double-stranded RNA molecules targeting the GPR87 gene were prepared and purified. 1.1B4 cancer cell lines were used as model pancreatic cancer cells. Produced siRNA molecules were encapsulated in Poly(Lactic-Co-Glycolic Acid) (PLGA) nano-micelles using three different methods, two of which were according to literature with (siR-PLGA-S) or without (siR-PLGA-V) sonication. However, a new method was suggested to overcome problems such as poly-dispersity and large sizes of siR-PLGA-S and siR-PLGA-V. The new method consists of encapsulating siRNA using mild agitation to the pre-made PLGA NPs. The latter method provided mono-dispersed particles (siR-P-PLGA) with 92 nm size and desired Encapsulation Efficiency (EE%). siR-P-PLGA was able to silence the GPR-87 gene in a ratio of 83.9%, almost 41 times more effective than siR-PLGA-S and siR-PLGA-V in HEK 293 T cells. siR-P-PLGA was able to show a mild cytotoxic effect on 1.1B4 pancreatic cancer cells within 48 h.


Assuntos
Marcação de Genes/métodos , Nanopartículas , Neoplasias Pancreáticas/genética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Interferente Pequeno/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Linhagem Celular Tumoral , Inativação Gênica/fisiologia , Engenharia Genética/métodos , Células HEK293 , Humanos , Nanopartículas/administração & dosagem , Neoplasias Pancreáticas/terapia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Renilla
6.
Carbohydr Polym ; 229: 115451, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826509

RESUMO

Oil-in-water nano-emulsions have been obtained in the HEPES 20 mM buffer solution / [Alkylamidoammonium:Kolliphor EL = 1:1] / [6 wt% ethylcellulose in ethyl acetate] system over a wide oil-to-surfactant range and above 35 wt% aqueous component at 25 °C. The nano-emulsion with an oil-to-surfactant ratio of 70/30 and 95 wt% aqueous component was used for nanoparticles preparation. These nanoparticles (mean diameter around 90 nm and zeta potential of +22 mV) were non-toxic to HeLa cells up to a concentration of 3 mM of cationic species. Successful complexation with an antisense phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was achieved at cationic/anionic charge ratios above 16, as confirmed by zeta potential measurements and an electrophoretic mobility shift assay, provided that no Fetal Bovine Serum is present in the cell culture medium. Importantly, Renilla luciferase gene inhibition shows an optimum efficiency (40%) for the cationic/anionic ratio 28, which makes these complexes promising for "in vitro" cell transfection.


Assuntos
Celulose/análogos & derivados , Nanopartículas/química , Oligonucleotídeos Antissenso/genética , Animais , Bovinos , Celulose/química , Celulose/toxicidade , Inativação Gênica , Técnicas de Transferência de Genes , Células HeLa , Humanos , Luciferases/antagonistas & inibidores , Luciferases/genética , Nanopartículas/toxicidade , RNA Mensageiro/genética , Renilla/enzimologia , Soroalbumina Bovina/química , Eletricidade Estática
7.
Arch Toxicol ; 93(11): 3141-3152, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31515601

RESUMO

The chemical warfare agent sulfur mustard (SM) alkylates a multitude of biomacromolecules including DNA and proteins. Cysteine residues and nucleophilic nitrogen atoms in purine DNA bases are typical targets of SM but potentially every nucleophilic structure may be alkylated by SM. In the present study, we analyzed potential SM-induced alkylation of glucocorticoid (GC) hormones and functional consequences thereof. Hydrocortisone (HC), the synthetic betamethasone (BM) and dexamethasone (DEX) were chosen as representative GCs. Structural modifications were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hypothesized alkylation was verified and structurally allocated to the OH-group of the C21 atom. The biological function of SM-alkylated GCs was investigated using GC-regulated dual-luciferase reporter gene assays and an ex vivo GC responsiveness assay coupled with real-time quantitative polymerase chain reaction (RT-qPCR). For the reporter gene assays, HEK293-cells were transiently transfected with a dual-luciferase reporter gene that is transcriptional regulated by a GC-response element. These cells were then incubated either with untreated or SM-derivatized HC, BM or DEX. Firefly-luciferase (Fluc) activity was determined 24 h after stimulation. Fluc-activity significantly decreased after stimulation with SM-pre-exposed GC dependent on the SM concentration. The ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to DEX revealed a transcriptional dysregulation of GC-regulated genes (FKBP5, IL1R2, and GILZ) after stimulation with SM-alkylated DEX. Our results present GCs as new biological targets of SM associated with a disturbance of hormone function.


Assuntos
Alquilantes/toxicidade , Substâncias para a Guerra Química/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Gás de Mostarda/toxicidade , Animais , Betametasona/farmacologia , Cotinina/análogos & derivados , Cotinina/farmacologia , Dexametasona/farmacologia , Genes Reporter , Glucocorticoides/genética , Células HEK293 , Humanos , Luciferases/genética , Renilla , Transfecção
8.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31511391

RESUMO

The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle.IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.


Assuntos
Hepacivirus/metabolismo , Serina/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Hepatite C/virologia , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Proteômica , Renilla , Proteínas não Estruturais Virais/química , Replicação Viral/fisiologia
9.
Gigascience ; 8(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30942866

RESUMO

BACKGROUND: More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral "forests," which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals. Here, we present a de novo assembly of an azooxanthellate shallow-water octocoral, Renilla muelleri. FINDINGS: We generated a hybrid de novo assembly using MaSuRCA v.3.2.6. The final assembly included 4,825 scaffolds and a haploid genome size of 172 megabases (Mb). A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome may be smaller (172 Mb minimum size) than other publicly available coral genomes (256-448 Mb), the R. muelleri genome is similar to other coral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models. CONCLUSIONS: The R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity.


Assuntos
Genoma , Genômica , Renilla/genética , Animais , Biologia Computacional/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular
10.
Nat Protoc ; 14(4): 1084-1107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911173

RESUMO

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Imagem Óptica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzenoacetamidas/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Pirazinas/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Renilla , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
13.
Anal Chem ; 90(21): 12986-12993, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30234965

RESUMO

Sensitive and selective quantification of individual sugars in complex media is technically challenging and usually requires HPLC separation. Accurate measurement without the need for separation would be highly desirable. The measurement of trace levels of lactose in lactose-reduced milk exemplifies the problem, with the added challenge that trace lactose must be measured in the presence of ≈140 mM glucose and galactose, the products of lactase digestion of lactose. Biosensing is an alternative to HPLC, but current biosensing methods, based on coupled-enzyme assays, tend to have poor sensitivity and complex biochemistry and can be time-consuming. We explored a fundamentally different approach, based on identifying a lactose-specific binding protein compatible with photonic transduction. We identified the BgaR transcriptional regulator of Clostridium perfringens, which is highly selective for lactose, as a suitable ligand binding domain and combined it with a bioluminescence energy resonance transfer transduction system. This BRET-based biosensor showed a 27% decrease in the BRET ratio in the presence of saturating (1 mM) lactose. Using a 5 min assay, the half maximal effective concentration (EC50) for lactose in phosphate-buffered saline (PBS) was 12 µM. The biosensor was 200 times more sensitive to lactose than to glucose or galactose. Sensitivity and selectivity were not significantly affected by the presence of 10% (v/v) dialyzed milk. The biosensor is suitable for direct determination of residual lactose in lactase-treated milk, with a limit of detection of 0.2 µM, 100 times below the most stringent lactose-free standard and without the need to remove fat or protein from the sample.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Lactose/análise , Leite/química , Fatores de Transcrição/química , Agrobacterium tumefaciens/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium perfringens/química , Transferência de Energia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lactose/metabolismo , Ligantes , Limite de Detecção , Luminescência , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Renilla/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Nat Commun ; 9(1): 3104, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082832

RESUMO

Dendritic cells use a specialized pathway called cross-presentation to activate CD8+ T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem. Here we describe the development of a sensitive reporter substrate, an N-glycosylated variant of Renilla luciferase fused to the Fc region of human IgG1. The luciferase variant is designed to be enzymatically inactive when glycosylated, but active after the asparagine to aspartic acid conversion that occurs upon deglycosylation by the cytosolic enzyme N-glycanase-1. The generation of cytosolic luminescence depends on internalization, deglycosylation, the cytosolic AAA-ATPase VCP/p97, and the cytosolic chaperone HSP90. By incorporating a T cell epitope into the fusion protein, we demonstrate that antigen dislocation into the cytosol is the rate limiting step in cross-presentation.


Assuntos
Linfócitos T CD8-Positivos/citologia , Apresentação Cruzada , Citosol/metabolismo , Imunoglobulina G/metabolismo , Animais , Apresentação do Antígeno , Antígenos/metabolismo , Células Dendríticas/imunologia , Endocitose , Epitopos/química , Glicosilação , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Luciferases/metabolismo , Chaperonas Moleculares/metabolismo , Ligação Proteica , Transporte Proteico , Renilla
15.
Chembiochem ; 19(13): 1409-1413, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29656613

RESUMO

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.


Assuntos
RNA Interferente Pequeno/química , Uridina/análogos & derivados , Animais , Ensaios Enzimáticos , Células HeLa , Humanos , Concentração Inibidora 50 , Luciferases de Renilla/genética , Estabilidade de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , Renilla/enzimologia , Estereoisomerismo , Temperatura , Uridina/química
16.
Protein Expr Purif ; 145: 39-44, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29288731

RESUMO

Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [1] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability. Furthermore, the mutant simultaneously improved a tolerance for protease digestion, e.g. trypsin and proteinase K, and for organic solvent. Residual activity of the mutant after treatment with 10% 2-propanol, 10% DMF and 20% DMSO at 35 °C for 1 h was 29.4, 24.8 and 91.3%, respectively, whereas that of the wild type was 0.4, 0.1 and 24.3%, respectively.


Assuntos
Temperatura Alta , Luciferases de Renilla/metabolismo , Mutagênese Sítio-Dirigida , Renilla/enzimologia , Animais , Estabilidade Enzimática , Luciferases de Renilla/química , Luciferases de Renilla/genética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Org Biomol Chem ; 15(48): 10238-10244, 2017 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-29177293

RESUMO

The prodrug or caged-luciferin strategy affords an excellent platform for persistent bioluminescence imaging. In the current work, we designed and synthesized ten novel pro-substrates for Renilla luciferase by introducing ester protecting groups of different sizes into the carbonyl group of the free luciferin 1. Taking advantage of intracellular esterases, lipases, and nucleophilic substances, the ester protecting groups were hydrolyzed, resulting in the release of a free luciferin and a bioluminescence signal turn-on. Among the tested pro-substrates, the butyryloxymethyl luciferin 7 exhibited low cytotoxicity and a prolonged luminescence signal both in cellulo and in vivo. Therefore, the butyryloxymethyl luciferin 7 can act as a promising substrate for noninvasive extended imaging in diagnostic and therapeutic fields.


Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/análise , Medições Luminescentes , Renilla/enzimologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/farmacologia , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Especificidade por Substrato
18.
Curr Protoc Protein Sci ; 90: 30.5.1-30.5.14, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29091275

RESUMO

The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed 'bimolecular complementation' relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins. When these proteins interact, they effectively reconstitute the activity of the molecular reporter (typically leading to increased fluorescence or luminescence). This unit describes the selection and development of bimolecular luminescence complementation (BiLC) assays for reporting intracellular PPIs, and provides examples in which BiLC was used to identify small molecules that can modulate PPIs. © 2017 by John Wiley & Sons, Inc.


Assuntos
Luciferases/genética , Medições Luminescentes/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Vaga-Lumes/química , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Luciferases/metabolismo , Luminescência , Medições Luminescentes/normas , Proteínas Recombinantes de Fusão/metabolismo , Renilla/química , Transfecção
19.
Int J Biol Macromol ; 105(Pt 1): 66-73, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28673845

RESUMO

Renilla luciferase (Rluc) from Renilla reniformis is an appropriate protein reporter for the detection of specific molecular targets due to its bioluminescent feature, although its relatively low stability limits the application. To investigate the effects of trehalose and sucrose as chemical chaperones on the kinetic stability of Rluc, we assayed the activity of the enzyme in the presence of these additives at high temperatures and to comprehend the mechanism of stability, molecular dynamic (MD) simulation was carried out. In the presence of trehalose a thermostabilizing effect which was considerable in comparison with other systems was observed. It is proposed that a wide radial like network of trehalose molecules supports α-helix structures that are located in the N-terminus and C-terminus of the protein. However, in the water simulation box, these helices alter to instable structures at high temperatures. Reduction of the fluctuation of these helices in the presence of trehalose molecules, may prevent the protein from unfolding and increase its shelf life.


Assuntos
Luciferases de Renilla/química , Luciferases de Renilla/metabolismo , Renilla/enzimologia , Temperatura , Trealose/química , Trealose/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Cinética , Modelos Moleculares , Conformação Proteica em alfa-Hélice/efeitos dos fármacos
20.
J Neuroinflammation ; 14(1): 111, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28577576

RESUMO

BACKGROUND: Sphingosine 1-phosphate (S1P) signals through G protein-coupled receptors to elicit a wide range of cellular responses. In CNS injury and disease, the blood-brain barrier is compromised, causing leakage of S1P from blood into the brain. S1P can also be locally generated through the enzyme sphingosine kinase-1 (Sphk1). Our previous studies demonstrated that S1P activates inflammation in murine astrocytes. The S1P1 receptor subtype has been most associated with CNS disease, particularly multiple sclerosis. S1P3 is most highly expressed and upregulated on astrocytes, however, thus we explored the involvement of this receptor in inflammatory astrocytic responses. METHODS: Astrocytes isolated from wild-type (WT) or S1P3 knockout (KO) mice were treated with S1P3 selective drugs or transfected with short interfering RNA to determine which receptor subtypes mediate S1P-stimulated inflammatory responses. Interleukin-6 (IL-6), and vascular endothelial growth factor A (VEGFa) messenger RNA (mRNA) and cyclooxygenase-2 (COX-2) mRNA and protein were assessed by q-PCR and Western blotting. Activation of RhoA was measured using SRE.L luciferase and RhoA implicated in S1P signaling by knockdown of Gα12/13 proteins or by inhibiting RhoA activation with C3 exoenzyme. Inflammation was simulated by in vitro scratch injury of cultured astrocytes. RESULTS: S1P3 was highly expressed in astrocytes and further upregulated in response to simulated inflammation. Studies using S1P3 knockdown and S1P3 KO astrocytes demonstrated that S1P3 mediates activation of RhoA and induction of COX-2, IL-6, and VEGFa mRNA, with some contribution from S1P2. S1P induces expression of all of these genes through coupling to the Gα12/13 proteins which activate RhoA. Studies using S1P3 selective agonists/antagonists as well as Fingolimod (FTY720) confirmed that stimulation of S1P3 induces COX-2 expression in astrocytes. Simulated inflammation increased expression of Sphk1 and consequently activated S1P3, demonstrating an autocrine pathway through which S1P is formed and released from astrocytes to regulate COX-2 expression. CONCLUSIONS: S1P3, through its ability to activate RhoA and its upregulation in astrocytes, plays a unique role in inducing inflammatory responses and should be considered as a potentially important therapeutic target for CNS disease progression.


Assuntos
Astrócitos/metabolismo , Expressão Gênica/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Lisoesfingolipídeo/genética , Renilla , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína rhoA de Ligação ao GTP/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...