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1.
Immunity ; 54(9): 2143-2158.e15, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34453881

RESUMO

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Encéfalo/patologia , COVID-19/imunologia , Pulmão/patologia , SARS-CoV-2/fisiologia , Testículo/patologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Encéfalo/virologia , COVID-19/terapia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Luciferases/genética , Medições Luminescentes , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Testículo/virologia
2.
Mol Cell Proteomics ; : 100856, 2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-39383947

RESUMO

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

3.
J Biol Chem ; 300(2): 105594, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38145744

RESUMO

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Melanoma , Humanos , Adenosina Trifosfatases/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/isolamento & purificação , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Melanoma/genética , Melanoma/fisiopatologia , Células HEK293
4.
J Biol Chem ; 299(9): 105107, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37517699

RESUMO

Protein-protein interactions (PPIs) form the foundation of any cell signaling network. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. However, incongruity may be observed across different PPI assays when investigating a protein partner of interest; these discrepancies can be partially attributed to the fusion of different large functional moieties, such as fluorescent proteins or enzymes, which can yield disparate perturbations to the protein's stability, subcellular localization, and interaction partners depending on the given cellular assay. Owing to their smaller size, epitope tags may exhibit a diminished susceptibility to instigate such perturbations. However, while they have been widely used for detecting or manipulating proteins in vitro, epitope tags lack the in vivo traceability and functionality needed for intracellular biosensors. Herein, we develop NbV5, an intracellular nanobody binding the V5-tag, which is suitable for use in cellular assays commonly used to study PPIs such as BRET, NanoBiT, and Tango. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling, specifically by replacing larger functional moieties attached to the protein interactors, such as fluorescent or luminescent proteins (∼30 kDa), by the significantly smaller V5-tag peptide (1.4 kDa), and for microscopy imaging which is successfully detected by NbV5-based biosensors. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs.

5.
Biochem Biophys Res Commun ; 733: 150711, 2024 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-39312880

RESUMO

Evaluating the steady-state protein level of the EGFR in live cells presents significant challenges compared to measuring its kinase activity. Traditional testing methods, such as immunoblotting, ELISA, and immunofluorescence assays, are generally restricted to fixed cells or cell lysates. Despite their utility, these methods are cumbersome and provide only intermittent snapshots of EGFR levels at specific time points. With emerging trends in drug development shifting toward engineering novel agents that promote protein degradation, rather than simply inhibiting kinase activity, a tool that enables real-time, quantitative detection of drug effects in live cells could catalyze advances in the field. Such an innovation would expedite the drug development process, enhancing the translation of research findings into effective, patient-centered therapies. The NanoLuc-EGFR cell line, created through CRISPR genome editing, allows for the continuous tracking and analysis of EGFR protein levels and their degradation within live cells. This approach provides quantitative monitoring of protein dynamics in real time, offering insights that go beyond absolute protein levels to include aspects such as protein stability and degradation rate. Using this cell line model, we observed that AT13387 and H84T BanLec induce EGFR degradation in A549-HiBiT cells, with the results confirmed by immunoblotting. In contrast, Erlotinib, Osimertinib, and Cetuximab inhibit EGFR phosphorylation without altering total EGFR levels, as validated by the HiBiT luciferase assay. The NanoLuc-EGFR cell line marks a significant advancement in understanding protein regulation and serves as an instrumental platform for investigating targeted therapies that modulate protein kinases, especially those that induce protein degradation.


Assuntos
Receptores ErbB , Receptores ErbB/metabolismo , Receptores ErbB/genética , Humanos , Linhagem Celular Tumoral , Edição de Genes , Compostos de Anilina/farmacologia , Acrilamidas/farmacologia , Proteólise , Linhagem Celular , Sistemas CRISPR-Cas , Indóis , Pirimidinas
6.
Anal Biochem ; 660: 114929, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36270332

RESUMO

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).


Assuntos
COVID-19 , Imunoglobulina G , Camundongos , Coelhos , Animais , Proteína Estafilocócica A , SARS-CoV-2 , Anticorpos Antivirais , Imunoensaio/métodos , Nucleoproteínas , Sensibilidade e Especificidade
7.
Mol Ther ; 30(9): 2998-3016, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-35526097

RESUMO

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).


Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Imidazóis , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Animais , Antivirais/farmacologia , Imidazóis/farmacologia , Camundongos , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Estados Unidos , United States Food and Drug Administration
8.
Exp Parasitol ; 254: 108620, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37716462

RESUMO

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.

9.
J Biol Chem ; 296: 100366, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33545176

RESUMO

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer-based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/química , Luciferases/metabolismo , Bioensaio , Membrana Celular/metabolismo , Transferência de Energia , Células HEK293 , Humanos , Cinética , Ligantes , Ligação Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
10.
J Cell Sci ; 133(1)2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31822628

RESUMO

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.


Assuntos
Expressão Gênica/genética , Peptídeo Hidrolases/metabolismo , Rhinovirus/genética , Humanos
11.
FEMS Yeast Res ; 21(8)2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34940882

RESUMO

The yeast two-hybrid (Y2H) assay is a powerful technique to identify protein-protein interactions. However, the auxotrophic markers that are the most common Y2H reporters take several days to yield data and require subjective assessment of semiquantitative data to identify interactions. Several reporters have been developed to overcome these disadvantages, but there is still a need for a Y2H reporter that is objective, fast and able to be performed with common laboratory equipment. In this report, we replaced the ADE2 reporter in BK100 with NanoLuc luciferase to yield BK100Nano. We developed an optimized assay to measure NanoLuc activity in 96-well plates and analyzed a set of 74 pairs identified in Y2H library screens, which revealed 44 positive interactions using an unbiased cutoff based on the mean luminescence of negative control samples. The same set was also tested for growth on Y2H selection medium via expression of the HIS3 reporter. We found 91% agreement between the two assays, with discrepancies attributed to weak interactions that displayed variable growth on Y2H medium. Overall, the new BK100Nano strain establishes a quantitative and convenient method to identify Y2H interactions and has potential to be applied to a high throughput manner.


Assuntos
Saccharomyces cerevisiae , Biblioteca Gênica , Genes Reporter , Luciferases/genética , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido
12.
Int J Mol Sci ; 23(7)2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35408820

RESUMO

The P2Y2 receptor (P2Y2R) is a G protein-coupled receptor that is activated by extracellular ATP and UTP, to a similar extent. This allows it to play roles in the cell's response to the (increased) release of these nucleotides, e.g., in response to stress situations, including mechanical stress and oxygen deprivation. However, despite its involvement in important (patho)physiological processes, the intracellular signaling induced by the P2Y2R remains incompletely described. Therefore, this study implemented a NanoBiT® functional complementation assay to shed more light on the recruitment of ß-arrestins (ßarr1 and ßarr2) upon receptor activation. More specifically, upon determination of the optimal configuration in this assay system, the effect of different (receptor) residues/regions on ßarr recruitment to the receptor in response to ATP or UTP was estimated. To this end, the linker was shortened, the C-terminal tail was truncated, and phosphorylatable residues in the third intracellular loop of the receptor were mutated, in either singly or multiply adapted constructs. The results showed that none of the introduced adaptations entirely abolished the recruitment of either ßarr, although EC50 values differed and time-luminescence profiles appeared to be qualitatively altered. The results hint at the C-terminal tail modulating the interaction with ßarr, while not being indispensable.


Assuntos
Trifosfato de Adenosina , Transdução de Sinais , Fosforilação , Uridina Trifosfato/farmacologia , beta-Arrestina 1 , beta-Arrestina 2/metabolismo , beta-Arrestinas
13.
Biochem Biophys Res Commun ; 534: 485-490, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33239166

RESUMO

To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Luciferases/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação/genética , COVID-19/metabolismo , COVID-19/virologia , Humanos , Luciferases/genética , Nanotecnologia/métodos , Ligação Proteica , Domínios Proteicos , Receptores Virais/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Tratamento Farmacológico da COVID-19
14.
Sens Actuators B Chem ; 3362021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35250176

RESUMO

Glypican-3 (GPC3) is a serological biomarker for the diagnosis of Hepatocellular carcinoma (HCC), but it is a challenging task to develop a bioassay for determination of the trace GPC3 in serum. In this study, Bioluminescense immunoassay based on bifunctional nanobody-nanoluciferase fusion was developed with the ultra-sensitive feature to achieve this goal. First, nanobodies special against GPC-3 binder as biological recognition element were generated by immunization and phage display technology. Second, The best clone GPN2 was fused with nanoluciferase as a dual-functional immunoreagent to establish an ultra-sensitive bioluminescence enzyme immunoassay (BLEIA), which is 30 and 5 times more sensitive than the traditional colorimetric assay and fluorescent assay, respectively. The cross-reactivity analysis of BLEIA showed that there was no cross-reactivity with HCC related tumor markers AFP, CEA, CA19-9 and GPC1/GPC2. The limit of detection (LOD) of developed BLEIA was 1.5 ng/mL, which assured its application in the diagnosis of GPC3 in 94 serum samples. This study indicates that BLEIA based on nanobody-nanoluciferase fusion could be used as a useful tool for the diagnosis of HCC patients.

15.
J Bacteriol ; 202(22)2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900827

RESUMO

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana/métodos , Micobacteriófagos/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/virologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia
16.
J Biol Chem ; 294(11): 4079-4090, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30593506

RESUMO

G protein-coupled receptors (GPCRs) are currently the target of more than 30% of the marketed medicines. However, there is an important medical need for ligands with improved pharmacological activities on validated drug targets. Moreover, most of these ligands remain poorly characterized, notably because of a lack of pharmacological tools. Thus, there is an important demand for innovative assays that can detect and drive the design of compounds with novel or improved pharmacological properties. In particular, a functional and screening-compatible GPCR-G protein interaction assay is still unavailable. Here, we report on a nanoluciferase-based complementation technique to detect ligands that promote a GPCR-G protein interaction. We demonstrate that our system can be used to profile compounds with regard to the G proteins they activate through a given GPCR. Furthermore, we established a proof of applicability of screening for distinct G proteins on dopamine receptor D2 whose differential coupling to Gαi/o family members has been extensively studied. In a D2-Gαi1versus D2-Gαo screening, we retrieved five agonists that are currently being used in antiparkinsonian medications. We determined that in this assay, piribedil and pergolide are full agonists for the recruitment of Gαi1 but are partial agonists for Gαo, that the agonist activity of ropinirole is biased in favor of Gαi1 recruitment, and that the agonist activity of apomorphine is biased for Gαo We propose that this newly developed assay could be used to develop molecules that selectively modulate a particular G protein pathway.


Assuntos
Luciferases/metabolismo , Nanopartículas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Ligantes , Luciferases/química , Nanopartículas/química , Pergolida/química , Pergolida/farmacologia , Piribedil/química , Piribedil/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química
17.
J Biol Chem ; 294(4): 1095-1103, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30559293

RESUMO

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.


Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/biossíntese , Ativação Enzimática/efeitos da radiação , Luminescência , Animais , Proliferação de Células , Células Cultivadas , Células HEK293 , Humanos , Luz , Luciferases/metabolismo , Ratos
18.
Retrovirology ; 17(1): 12, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32430080

RESUMO

BACKGROUND: The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. RESULTS: The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. CONCLUSION: These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.


Assuntos
Vetores Genéticos , HIV-1/genética , HIV-1/fisiologia , Medições Luminescentes/métodos , Liberação de Vírus , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Células HeLa , Humanos , Luciferases , Mutação , Sensibilidade e Especificidade , Vírion/metabolismo , Montagem de Vírus
19.
BMC Infect Dis ; 20(1): 906, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33256616

RESUMO

BACKGROUNDS: Primary infection with Toxoplasma gondii during pregnancy can pose serious health problems for the fetus. However, the epidemiological status of toxoplasmosis among reproductive-aged population in Myanmar is largely unknown. Although luciferase immunoprecipitation system (LIPS) assays for serodiagnosis of toxoplasmosis was developed mostly using mouse infection model, had not been tested by using field-derived human samples. METHODS: A total of 251 serum samples were collected from reproductive-aged women, residing in Shwegyin township, Bago region, Myanmar and analyzed with a commercial ELISA kit, as well as in-house LIPS assays. RESULTS: The overall seroprevalence for Toxoplasma gondii infection by the commercial ELISA was 11.5%. No clear risk factor was identified except for being in the younger age group (15-30 years old). Overall, LIPS assays showed low sensitivity when the commercial ELSA was used as a reference test. CONCLUSION: We identified the epidemiological situation of toxoplasmosis in some rural communities in Myanmar. The data obtained here will serve as a primary information for the effort to reduce toxoplasmosis in this region. Although looked promising in the previous experiments with mouse infection model, we found that the reported LIPS procedures need further improvements to increase the sensitivities.


Assuntos
Imunoprecipitação/métodos , Medições Luminescentes/métodos , Testes Sorológicos/métodos , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Toxoplasmose/epidemiologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Luciferases , Substâncias Luminescentes , Camundongos , Pessoa de Meia-Idade , Mianmar/epidemiologia , Fatores de Risco , População Rural , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Toxoplasmose/sangue , Toxoplasmose/parasitologia , Adulto Jovem
20.
Anal Bioanal Chem ; 412(29): 8015-8022, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32926202

RESUMO

The Mu opioid receptor (MOR) has been the subject of intense research over the past decades, especially in the field of analgesic therapeutics. It is the primary target for both clinical and recreational opioids. Recently, camelid-derived nanobodies have received significant attention due to their applicability in stabilizing the crystal structure of activated MOR, via specific recognition of and binding to the active receptor conformation. In the present study, we developed and applied a novel bio-assay to monitor MOR activation, utilizing intracellular expression of one such nanobody, Nb39. The principle of functional complementation of a split nanoluciferase was used to assess recruitment of Nb39 to MOR, following activation by a set of five synthetic opioids. The obtained pharmacological parameters-negative logarithm of EC50 (pEC50, as a measure of potency) and maximal response provoked by a ligand (Emax, as a measure of efficacy; relative to hydromorphone)-were compared with those obtained using a G protein recruitment assay, in which a mini-Gi protein (engineered GTPase domain of Gαi subunit) is recruited to activated MOR. Similar EC50 but distinct Emax values were obtained with both bio-assays, with lower Emax values for the Nb-based bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR. Graphical abstract.


Assuntos
Luciferases/metabolismo , Receptores Opioides mu/metabolismo , Anticorpos de Domínio Único , Analgésicos Opioides/farmacologia , Bioensaio , Células HEK293 , Humanos , Luciferases/química , Tamanho da Partícula , Receptores Opioides mu/agonistas
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