Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Nat Chem Biol ; 18(6): 596-604, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35314814

RESUMO

Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.


Assuntos
Mieloma Múltiplo , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Ligantes , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética
2.
J Immunol ; 200(5): 1937-1950, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29351998

RESUMO

Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab-anti-idiotype complexes, and anti-trinitrophenol-trinitrophenol complexes in a sensitive manner (≤1 µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/imunologia , Receptores de IgG/imunologia , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Linhagem Celular , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fosfoproteínas/imunologia , Rituximab/imunologia , Transdução de Sinais/imunologia , Domínios de Homologia de src/imunologia
3.
Blood ; 126(8): e1-e10, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26124495

RESUMO

Growing interest in natural killer (NK) cell-based therapy for treating human cancer has made it imperative to develop new tools to measure early events in cell death. We recently demonstrated that protease-cleavable luciferase biosensors detect granzyme B and pro-apoptotic caspase activation within minutes of target cell recognition by murine cytotoxic lymphocytes. Here we report successful adaptation of the biosensor technology to assess perforin-dependent and -independent induction of death pathways in tumor cells recognized by human NK cell lines and primary cells. Cell-cell signaling via both Fc receptors and NK-activating receptors led to measurable luciferase signal within 15 minutes. In addition to the previously described aspartase-cleavable biosensors, we report development of granzyme A and granzyme K biosensors, for which no other functional reporters are available. The strength of signaling for granzyme biosensors was dependent on perforin expression in IL-2-activated NK effectors. Perforin-independent induction of apoptotic caspases was mediated by death receptor ligation and was detectable after 45 minutes of conjugation. Evidence of both FasL and TRAIL-mediated signaling was seen after engagement of Jurkat cells by perforin-deficient human cytotoxic lymphocytes. Although K562 cells have been reported to be insensitive to TRAIL, robust activation of pro-apoptotic caspases by NK cell-derived TRAIL was detectable in K562 cells. These studies highlight the sensitivity of protease-cleaved luciferase biosensors to measure previously undetectable events in live cells in real time. Further development of caspase and granzyme biosensors will allow interrogation of additional features of granzyme activity in live cells including localization, timing, and specificity.


Assuntos
Apoptose/fisiologia , Técnicas Biossensoriais , Granzimas/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Caspases/imunologia , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , Citometria de Fluxo , Granzimas/administração & dosagem , Humanos , Immunoblotting , Células Jurkat , Células K562 , Proteínas Recombinantes , Transfecção
4.
Chemistry ; 22(30): 10369-75, 2016 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-27305599

RESUMO

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems.

5.
J Immunol ; 193(2): 519-28, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24928990

RESUMO

Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a (51)Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B- compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B-deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs.


Assuntos
Caspases/imunologia , Granzimas/imunologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/imunologia , Animais , Apoptose/imunologia , Sítios de Ligação/genética , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/imunologia , Caspase 7/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Ativação Enzimática/imunologia , Granzimas/genética , Granzimas/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Perforina/genética , Perforina/imunologia , Perforina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo , Receptor fas/metabolismo
6.
Anal Biochem ; 489: 1-8, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26278171

RESUMO

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.


Assuntos
Proteínas de Artrópodes/metabolismo , Endocitose , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Descoberta de Drogas/métodos , Endocitose/efeitos dos fármacos , Corantes Fluorescentes/química , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Ligantes , Luciferases/química , Luciferases/genética , Microscopia Confocal , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
7.
Wellcome Open Res ; 5: 20, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32587898

RESUMO

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â ® Binary Technology (NanoBiT â ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

8.
J Mol Biol ; 365(4): 945-57, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17116308

RESUMO

In an effort to extend the peptide aptamer approach, we have developed a scaffold protein that allows small molecule ligand control over the presentation of a peptide aptamer. This scaffold, a fusion of three protein domains, FKBP12, FRB, and GST, presents a peptide linker region for target protein binding only in the absence of the small molecule Rapamycin or other non-immunosuppressive Rapamycin derivatives. Here we describe the characterization of ligand-regulated peptide aptamers that interact with and inhibit the 5'-AMP-activated protein kinase (AMPK). AMPK, a central regulator of cellular energy homeostasis, responds to high cellular AMP/ATP ratios by promoting energy producing pathways and inhibiting energy consuming biosynthetic pathways. We have characterized 15 LiRPs of similar, poly-basic sequence and have determined that they interact with the substrate peptide binding region of both AMPK alpha1 and alpha2. These proteins, some of which serve as poor substrates of AMPK, inhibit the kinase as pseudosubstrates in a Rapamycin-regulated fashion in vitro, an effect that is largely competitive with substrate peptide and mediated by an increase in the kinase's apparent K(m) for substrate peptide. This pseudosubstrate inhibition of AMPK by LiRP proteins reduced the AMP stimulation of AMPK in vitro and caused the inhibited state of the kinase to kinetically resemble the basal, unstimulated state of AMPK, providing potential insight into the molecular mechanisms of AMP stimulation of AMPK.


Assuntos
Aptâmeros de Peptídeos/química , Complexos Multienzimáticos/química , Peptídeos/química , Proteínas Serina-Treonina Quinases/química , Proteínas Quinases Ativadas por AMP , Sequência de Aminoácidos , Animais , Glutationa Transferase/metabolismo , Imunossupressores/farmacologia , Cinética , Ligantes , Conformação Molecular , Dados de Sequência Molecular , Fosforilação , Ratos , Homologia de Sequência de Aminoácidos , Sirolimo/química
9.
ACS Chem Biol ; 13(2): 467-474, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28892606

RESUMO

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Proteínas Luminescentes/genética , Oligopeptídeos/genética , Proteínas Adaptadoras de Transdução de Sinal , Anticorpos/química , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Proteína 9 Associada à CRISPR/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Genes Reporter/genética , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leupeptinas/farmacologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Luciferases/metabolismo , Luminescência , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Streptococcus pyogenes/enzimologia
10.
Nucleic Acids Res ; 33(6): e55, 2005 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-15800206

RESUMO

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1-3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from approximately 60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of approximately 1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.


Assuntos
Embaralhamento de DNA/métodos , Genes Sintéticos , Oligodesoxirribonucleotídeos/síntese química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Modelos Teóricos , Proteína MutS de Ligação de DNA com Erro de Pareamento , Mutação , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Reação em Cadeia da Polimerase
11.
Org Lett ; 8(11): 2357-60, 2006 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-16706525

RESUMO

[reaction: see text] We report the development of a safety-catch photolabile linker that allows the light-directed synthesis and spatially selective photorelease of oligonucleotides from microarrays. The linker remains stable to light during DNA synthesis, and is activated for photorelease after acidic hydrolysis. We demonstrate that the photoreleased oligonucleotides can be amplified by PCR to produce double stranded DNA. The advantages offered by this linker could aid the development of an automated gene synthesis platform.


Assuntos
DNA/síntese química , Oligonucleotídeos/síntese química , Fotólise , DNA/química , Estrutura Molecular , Oligonucleotídeos/química
12.
Chem Biol ; 12(7): 847-55, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16039531

RESUMO

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.


Assuntos
Glutationa Transferase/metabolismo , Peptídeos/farmacologia , Proteína do Retinoblastoma/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas Quinases Ativadas por AMP , Sequência de Aminoácidos , Sítios de Ligação , Células Cultivadas , Interações Medicamentosas , Ligantes , Complexos Multienzimáticos/metabolismo , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteína do Retinoblastoma/genética , Proteínas de Ligação a Tacrolimo/genética
13.
ACS Chem Biol ; 11(2): 400-8, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26569370

RESUMO

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 µM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 ß-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and ß-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Arrestinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Medições Luminescentes/métodos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , beta-Arrestina 2 , beta-Arrestinas , beta-Lactamases/metabolismo
14.
PLoS One ; 8(6): e66248, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23776643

RESUMO

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.


Assuntos
Caspases/metabolismo , Medições Luminescentes/métodos , Animais , Apoptose/fisiologia , Técnicas Biossensoriais , Western Blotting , Linhagem Celular Tumoral , Humanos , Camundongos , Peptídeo Hidrolases/metabolismo
15.
ACS Chem Biol ; 7(11): 1848-57, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22894855

RESUMO

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.


Assuntos
Crustáceos/enzimologia , Genes Reporter , Luciferases/análise , Luciferases/genética , Engenharia de Proteínas , Pirazinas/metabolismo , Animais , Linhagem Celular , Crustáceos/química , Crustáceos/genética , Crustáceos/metabolismo , Estabilidade Enzimática , Vaga-Lumes/enzimologia , Expressão Gênica , Humanos , Luciferases/metabolismo , Substâncias Luminescentes/análise , Substâncias Luminescentes/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Renilla/enzimologia , Temperatura
16.
Methods Mol Biol ; 756: 263-71, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21870231

RESUMO

G-protein coupled, seven-transmembrane (7-TM) receptors (GPCRs) comprise a diverse class of signaling molecules involved in cellular physiology and pathology. In recent years, intracellular biosensors have been developed to monitor changes in intracellular cAMP in real time, facilitating studies on the mechanisms of GPCR activation and desensitization in living cells. However, methods based on fluorescence can show limitations in response dynamics together with being difficult to perform. Here we present the use of genetically encoded, luminescent biosensors that allow a facile, non-lytic assay format for monitoring cAMP dynamics in living cells.


Assuntos
Técnicas Biossensoriais/métodos , AMP Cíclico/análise , Medições Luminescentes/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Vaga-Lumes/enzimologia , Humanos , Luciferases/análise , Luciferases/genética , Substâncias Luminescentes/análise , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Transfecção
17.
ACS Chem Biol ; 6(11): 1193-7, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21932825

RESUMO

The second messenger cAMP is a key mediator of signal transduction following activation of G-protein coupled receptors. Investigations on Gs-coupled receptors would benefit from a second messenger assay that allows continuous monitoring of kinetic changes in cAMP concentration over a broad dynamic range. To accomplish this, we have evolved a luminescent biosensor for cAMP to better encompass the physiological concentration ranges present in living cells. When compared to an immunoassay, the evolved biosensor construct was able to accurately track both the magnitude and kinetics of cAMP change using a far less labor intensive format. We demonstrate the utility of this construct to detect a broad range of receptor activity, together with showing suitability for use in high-throughput screening.


Assuntos
Técnicas Biossensoriais/métodos , AMP Cíclico/análise , Ensaios de Triagem em Larga Escala/métodos , Medições Luminescentes , AMP Cíclico/química , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Termodinâmica
18.
ACS Chem Biol ; 3(6): 346-51, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18570354

RESUMO

Genetically encoded biosensors have proven valuable for real-time monitoring of intracellular phenomena, particularly FRET-based sensors incorporating variants of green fluorescent protein. To increase detection sensitivity and response dynamics, we genetically engineered firefly luciferase to detect specific intermolecular interactions through modulation of its luminescence activity. This concept has been applied in covalent, noncovalent, and allosteric design configurations. The covalent design gives sensitive detection of protease activity through a cleavage-dependent increase in luminescence. The noncovalent and allosteric designs allow reversible detection of the small molecules rapamycin and cAMP, respectively. These sensors allow detection of molecular processes within living cells following addition of the luciferin substrate to the growth medium. For example, the cAMP sensor allows monitoring of intracellular signal transduction associated with G-protein coupled receptor function. These and other luminescent biosensors will be useful for the sensitive detection of cellular physiology in research and drug discovery.


Assuntos
Técnicas Biossensoriais/métodos , Células/citologia , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Código Genético , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Regulação Alostérica , Células/metabolismo , Células Cultivadas , AMP Cíclico/análise , AMP Cíclico/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Humanos , Luciferases de Vaga-Lume/química , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais , Sirolimo/análise , Sirolimo/metabolismo
19.
Chemistry ; 10(17): 4334-40, 2004 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-15352116

RESUMO

We describe the first solid-phase synthesis of dihydrovirginiamycin S(1), a member of the streptogramin B family of antibiotics, which are nonribosomal-peptide natural products produced by Streptomyces. These compounds, along with the synergistic group A components, are "last line of defense" antimicrobial agents for the treatment of life-threatening infections such as vancomycin-resistant enterococci. The synthesis features an on-resin cyclization and is designed to allow production of streptogramin B analogues with diversification at positions 1', 1, 2, 3, 4, and 6. Several synthetic challenges known to hinder the synthesis of this class of compounds were solved, including sensitivity to acids and bases, and epimerization and rearrangements, through the judicious choice of deprotection conditions, coupling conditions, and synthetic strategy. This work should enable a better understanding of structure-activity relationships in the streptogramin B compounds, possible identification of analogues that bypass known resistance mechanisms, and perhaps the identification of analogues with novel biological activities.


Assuntos
Antibacterianos/síntese química , Estreptogramina B/síntese química , Estreptogramina Grupo B/síntese química , Virginiamicina/síntese química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Ciclização , Indicadores e Reagentes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estreptogramina B/análogos & derivados , Estreptogramina B/farmacologia , Estreptogramina Grupo B/farmacologia , Relação Estrutura-Atividade , Virginiamicina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA