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1.
BMC Cancer ; 21(1): 1074, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34598688

RESUMO

BACKGROUND: The human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC). METHODS: RT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression. RESULTS: In the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3'UTR. CONCLUSION: These findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.


Assuntos
Ciclina G2/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias de Cabeça e Pescoço/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Regiões 3' não Traduzidas/genética , Apoptose/genética , Área Sob a Curva , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ciclina G2/genética , Regulação para Baixo , Feminino , Inativação Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Luciferases/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Transfecção , Regulação para Cima
2.
Sci Rep ; 11(1): 18428, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531417

RESUMO

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/análise , COVID-19/prevenção & controle , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais/análise , Brasil , COVID-19/imunologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Estados Unidos , Vacinação
3.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443682

RESUMO

Atherosclerosis is the main cause of cardiovascular diseases which in turn, lead to the highest number of mortalities globally. This pathophysiological condition is developed due to a constant elevated level of plasma cholesterols. Statin is currently the widely used treatment in reducing the level of cholesterols, however, it may cause adverse side effects. Therefore, there is an urgent need to search for new alternative treatment. PCSK9 is an enzyme responsible in directing LDL-receptor (LDL-R)/LDL-cholesterols (LDL-C) complex to lysosomal degradation, preventing the receptor from recycling back to the surface of liver cells. Therefore, PCSK9 offers a potential target to search for small molecule inhibitors which inhibit the function of this enzyme. In this study, a marine invertebrate Acanthaster planci, was used to investigate its potential in inhibiting PCSK9 and lowering the levels of cholesterols. Cytotoxicity activity of A. planci on human liver HepG2 cells was carried out using the MTS assay. It was found that methanolic extract and fractions did not exhibit cytotoxicity effect on HepG2 cell line with IC50 values of more than 30 µg/mL. A compound deoxythymidine also did not exert any cytotoxicity activity with IC50 value of more than 4 µg/mL. Transient transfection and luciferase assay were conducted to determine the effects of A. planci on the transcriptional activity of PCSK9 promoter. Methanolic extract and Fraction 2 (EF2) produced the lowest reduction in PCSK9 promoter activity to 70 and 20% of control at 12.5 and 6.25 µg/mL, respectively. In addition, deoxythymidine also decreased PCSK9 promoter activity to the lowest level of 60% control at 3.13 µM. An in vivo study using Sprague Dawley rats demonstrated that 50 and 100 mg/kg of A. planci methanolic extract reduced the total cholesterols and LDL-C levels to almost similar levels of untreated controls. The level of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) showed that the administration of the extract did not produce any toxicity effect and cause any damage to rat liver. The results strongly indicate that A. planci produced a significant inhibitory activity on PCSK9 gene expression in HepG2 cells which may be responsible for inducing the uptake of cholesterols by liver, thus, reducing the circulating levels of total cholesterols and LDL-C. Interestingly, A. planci also did show any adverse hepato-cytotoxicity and toxic effects on liver. Thus, this study strongly suggests that A. planci has a vast potential to be further developed as a new class of therapeutic agent in lowering the blood cholesterols and reducing the progression of atherosclerosis.


Assuntos
Colesterol/sangue , Pró-Proteína Convertase 9/antagonistas & inibidores , Estrelas-do-Mar/química , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Morte Celular , Proliferação de Células , LDL-Colesterol/sangue , Células Hep G2 , Humanos , Luciferases/metabolismo , Masculino , Metanol , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ratos Sprague-Dawley , Timidina/farmacologia , Triglicerídeos/sangue
4.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445534

RESUMO

Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis.


Assuntos
Glicerol/metabolismo , Luciferases/química , Luciferases/metabolismo , Modelos Teóricos , Photobacterium/enzimologia , Sacarose/metabolismo , Catálise , Domínio Catalítico , Difusão , Simulação de Dinâmica Molecular , Viscosidade
5.
Am J Physiol Cell Physiol ; 321(3): C443-C452, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34260301

RESUMO

Ventricular septal defects (VSDs) are the most common congenital heart defects (CHDs). Studies have documented that ISL1 has a crucial impact on cardiac growth, but the role of variants in the ISL1 gene promoter in patients with VSD has not been explored. In 400 subjects (200 patients with isolated and sporadic VSDs: 200 healthy controls), we investigated the ISL1 gene promoter variant and performed cellular functional experiments by using the dual-luciferase reporter assay to verify the impact on gene expression. In the ISL1 promoter, five variants were found only in patients with VSD by sequencing. Cellular functional experiments demonstrated that three variants decreased the transcriptional activity of the ISL1 promoter (P < 0.05). Further analysis with the online JASPAR database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants, possibly resulting in change of ISL1 protein expression and VSD formation. Our study has, for the first time, identified novel variants in the ISL1 gene promoter region in the Han Chinese patients with isolated and sporadic VSD. In addition, the cellular functional experiments, electrophoretic mobility shift assay, and bioinformatic analysis have demonstrated that these variants significantly alter the expression of the ISL1 gene and affect the binding of transcription factors, likely resulting in VSD. Therefore, this study may provide new insights into the role of the gene promoter region for a better understanding of genetic basis of the formation of CHDs and may promote further investigations on mechanism of the formation of CHDs.


Assuntos
Comunicação Interventricular/genética , Proteínas com Homeodomínio LIM/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Adolescente , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Sítios de Ligação , Estudos de Casos e Controles , Criança , Pré-Escolar , Bases de Dados Genéticas , Feminino , Expressão Gênica , Genes Reporter , Células HEK293 , Comunicação Interventricular/etnologia , Comunicação Interventricular/metabolismo , Comunicação Interventricular/patologia , Humanos , Lactente , Proteínas com Homeodomínio LIM/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Ligação Proteica , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Septo Interventricular/metabolismo , Septo Interventricular/patologia
6.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200322

RESUMO

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.


Assuntos
Benzodioxóis/farmacologia , Benzotiazóis/metabolismo , Bioensaio/métodos , Luciferases/metabolismo , Luminescência , Monoacilglicerol Lipases/metabolismo , Piperidinas/farmacologia , Ansiolíticos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Monoacilglicerol Lipases/antagonistas & inibidores
7.
Nat Commun ; 12(1): 4586, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321486

RESUMO

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/normas , Medições Luminescentes/normas , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/sangue , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/instrumentação , Calibragem , Proteínas de Ligação ao GTP/química , Genes Reporter , Humanos , Imunoconjugados/química , Limite de Detecção , Luciferases/genética , Luciferases/metabolismo , Testes Imediatos , SARS-CoV-2/genética
8.
Methods Mol Biol ; 2268: 137-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085266

RESUMO

Here we describe the stepwise application of bioluminescence resonance energy transfer (BRET)-based conformational receptor biosensors to study GPCR activation in intact cells. This technology can be easily adopted to various plate reader devices and microtiter plate formats. Due to the high sensitivity of these BRET-based receptor biosensors and their ability to quantify simultaneously receptor activation/de-activation kinetics as well as compound efficacy and potency, these optical tools provide the most direct and unbiased approach to monitor GPCR activity in a high-throughput-compatible assay format, representing a novel promising tool for the discovery of potential GPCR therapeutics.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Conformação Proteica , Receptores Acoplados a Proteínas G/química
9.
Methods Mol Biol ; 2268: 149-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085267

RESUMO

G protein-coupled receptors (GPCR) are one of the principal class of membrane proteins and around 30% of the currently marketed drugs act on one of them. The efficacious detection of ligands with the desired pharmacological profile remains a challenge of paramount importance in the GPCR drug discovery and pharmacological research. Recent evidences demonstrate that GPCR ligands can stabilize distinct receptor conformation and trigger various signaling pathways with different efficacies and/or potencies. This phenomenon called functional selectivity or biased signaling may lead to improved drugs with fewer side effects. Most receptors are promiscuous and can couple to more than one G protein family. To enable the discovery of biased ligands able to selectively trigger one G protein pathway over another, simple and efficient screening procedures are needed. The traditional assays aiming at detecting G protein activation monitor the generation of second messengers ([Ca2+]i, cAMP, IP1) or active G proteins (with GTP-g-S for instance). While these approaches have proven sensitive and robust, they are not suited for the detection of a single GPCR-G protein interaction. Here, we present in detail a method to assess directly the interaction between the receptor and the G protein. It permits the profiling of a receptor or a ligand toward G protein interactions and is compatible with high-throughput screening.


Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Proteínas de Ligação ao GTP/metabolismo , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
10.
Methods Mol Biol ; 2268: 233-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085273

RESUMO

Cytosolic ß-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or ß-arrestin recruitment makes ß-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor ß-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/metabolismo , Análise de Célula Única/métodos , beta-Arrestina 2/metabolismo , Células HEK293 , Humanos , Ligantes , Imagem Molecular/métodos , Ligação Proteica , Transdução de Sinais
11.
Methods Mol Biol ; 2268: 249-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085274

RESUMO

An understanding of the kinetic contributions to G protein-coupled receptor pharmacology and signaling is increasingly important in compound profiling. Nonequilibrium conditions are commonly present in vivo, for example, as the drug competes with dynamic changes in hormone or neurotransmitter concentration for the receptor. Under such conditions individual binding kinetic properties of the ligands can influence duration of action, local ligand concentration, and functional properties such as the degree of insurmountable inhibition. Mapping the kinetic patterns of GPCR signaling events elicited by agonists, rather than a peak response at a single timepoint, is often key to predicting their functional impact. This is also a path to a better understanding of the origins of ligand bias, and whether such ligands demonstrate their effects through selection of distinct GPCR conformations, or via their kinetic properties. Recent developments in complementation approaches, based on a small bright shrimp luciferase Nanoluc, provide a new route to kinetic analysis of GPCR signaling in living cells that is amenable to the throughput required for compound profiling. In the NanoBiT luciferase complementation system, GPCRs and effector proteins are tagged with Nanoluc fragments optimized for their low interacting affinity and stability. The interactions brought about by GPCR recruitment of the effector are reproduced by a rapid and reversible increase in NanoBiT luminescence, in the presence of its substrate furimazine. Here we discuss the methods for optimizing and validating the GPCR NanoBiT assays, and protocols for their application to study endpoint and kinetic aspects of agonist and antagonist pharmacology. We also describe how timecourse families of agonist concentration response curves, derived from a single NanoBiT assay experiment, can be used to evaluate the kinetic components in operational model derived parameters of ligand bias.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Imagem Molecular/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Células HEK293 , Humanos , Cinética , Ligantes , Luminescência , Transdução de Sinais
12.
Cancer Sci ; 112(9): 3484-3490, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34187084

RESUMO

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/farmacocinética , Interferon gama/deficiência , Interferon gama/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/metabolismo , Neoplasias Cutâneas/patologia , Vacinação/métodos
13.
J Biol Chem ; 297(1): 100885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34146545

RESUMO

PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus-infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2-induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.


Assuntos
Infecções por Orthomyxoviridae/metabolismo , Multimerização Proteica , Respiração , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Luciferases/genética , Luciferases/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Infecções por Orthomyxoviridae/fisiopatologia , Infecções por Orthomyxoviridae/virologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Proteínas Virais/genética
14.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065854

RESUMO

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.


Assuntos
Ligação Competitiva , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Aprendizado de Máquina , Receptores Acoplados a Proteínas G/metabolismo , Descoberta de Drogas/métodos , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Transfecção
15.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065887

RESUMO

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Genes Reporter , Animais , Animais Geneticamente Modificados , Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Drosophila melanogaster , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética , Luciferases/metabolismo , Oxaliplatina/administração & dosagem , Oxaliplatina/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066779

RESUMO

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.


Assuntos
Barreira Hematoencefálica/metabolismo , Leptina/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Animais , Sítios de Ligação , Composição Corporal , Peso Corporal , Células CHO , Plexo Corióideo/metabolismo , Cricetulus , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Luciferases/metabolismo , Masculino , Modelos Biológicos , Fosforilação , Transporte Proteico , Receptores para Leptina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Suínos
17.
Methods Mol Biol ; 2324: 131-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165713

RESUMO

Pseudogenes may function as competitive endogenous RNAs (ceRNAs), where they regulate the expression of genes by sequestering shared miRNAs. ceRNAs are becoming more extensively identified and studied, and demonstrating the dependence of their effects on miRNA sequestration is critical to establish them as ceRNAs. Here, we outline an experimental approach to assess the miRNA dependency of a candidate pseudogene ceRNA.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Pseudogenes/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Animais , Linhagem Celular , Reagentes para Ligações Cruzadas , Genes Reporter/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transcrição Genética , Transfecção
18.
Toxins (Basel) ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065185

RESUMO

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences.


Assuntos
Anti-Infecciosos/farmacologia , Histidina/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Anti-Infecciosos/química , Linhagem Celular Tumoral , Desenho de Fármacos , Fluoresceínas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luciferases/metabolismo , Peptídeos/química , Saporinas/metabolismo
19.
Methods Mol Biol ; 2268: 77-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085262

RESUMO

More than 30% of all pharmaceuticals target G-protein-coupled receptors (GPCRs). Here, we present a GPCR-based screen in yeast to identify ligands for human serotonin receptor 4 (5-HTR4). Serotonin receptor 4 agonists are used for the treatment of irritable bowel syndrome with constipation. Specifically, the HTR4-based screen couples activation of 5-HTR4 on the yeast cell surface to luciferase reporter expression. The HTR4-based screen has a throughput of one compound per second allowing the screening of more than a thousand compounds per day.


Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores 5-HT4 de Serotonina/química , Saccharomyces cerevisiae/metabolismo , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Genes Reporter , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligantes , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Saccharomyces cerevisiae/genética
20.
Nat Commun ; 12(1): 3616, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127663

RESUMO

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.


Assuntos
Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Animais , Sítios de Ligação , Catálise , Estabilidade Enzimática , Cinética , Luciferases de Renilla/química , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Mamíferos , Camundongos , Mutagênese , Mutação , Células NIH 3T3 , Conformação Proteica , Temperatura
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