RESUMEN
The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.
Asunto(s)
Hipoxia de la Célula , Quinasa 8 Dependiente de Ciclina/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Complejo Mediador/metabolismo , Neoplasias/metabolismo , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Acetilación , Línea Celular Tumoral , Quinasa 8 Dependiente de Ciclina/química , Quinasas Ciclina-Dependientes/metabolismo , Células HeLa , Histonas/metabolismo , HumanosRESUMEN
RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.
Asunto(s)
Algoritmos , Anotación de Secuencia Molecular , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/clasificación , ARN/química , Animales , Sitios de Unión , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expresión Génica , Ontología de Genes , Células HEK293 , Humanos , Motivos de Nucleótidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Programas Informáticos , Dedos de ZincRESUMEN
Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.
Asunto(s)
Bioensayo/métodos , Proteínas Cullin/metabolismo , Proteína NEDD8/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Cullin/genética , Células HCT116 , Células HEK293 , Humanos , Proteína NEDD8/genéticaRESUMEN
TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismo , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética , 5-Metilcitosina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Islas de CpG , Citosina/análogos & derivados , Citosina/metabolismo , Epigénesis Genética , Glicosilación , Histonas/metabolismo , Factor C1 de la Célula Huésped/metabolismo , Humanos , Inmunoprecipitación , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genéticaRESUMEN
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Proteínas Quinasas/genética , Reparación del ADN/genética , ADN/genéticaRESUMEN
In the trabecular meshwork (TM) of the eye, regulation of tissue contractility by the PPRARI sequence within the Heparin II (HepII) domain of fibronectin is believed to control the movement of aqueous humor and dictate the level of intraocular pressure. This study shows that the HepII domain utilizes activated alpha4beta1 integrin and collagen to mediate a co-signaling pathway that down-regulates contractility in TM cells. siRNA silencing of alpha4beta1 integrin blocked the actin disrupting effects of both PPRARI and the HepII domain. The down-regulation of the actin cytoskeleton and contractility did not involve syndecan-4 or other heparan sulfate proteoglycans (HSPGs) since siRNA silencing of syndecan-4 expression or heparitinase removal of cell surface HSPGs did not prevent the HepII-mediated disruption of the actin cytoskeleton. HepII-mediated disruption of the cytoskeleton depended upon the presence of collagen in the extracellular matrix, and cell binding studies indicated that HepII signaling involved cross-talk between alpha4beta1 and alpha1/alpha2beta1 integrins. This is the first time that the PPRARI sequence in the HepII domain has been shown to serve as a physiological alpha4beta1 ligand, suggesting that alpha4beta1 integrin may be a key regulator of tissue contractility.
Asunto(s)
Colágeno/metabolismo , Fibronectinas/metabolismo , Heparina/metabolismo , Integrina alfa4beta1/metabolismo , Presión Intraocular , Transducción de Señal , Malla Trabecular/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Adhesión Celular , Células Cultivadas , Citoesqueleto/metabolismo , Regulación hacia Abajo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Integrina alfa4beta1/antagonistas & inhibidores , Integrina alfa4beta1/genética , Macaca mulatta , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Malla Trabecular/citologíaRESUMEN
We report a small-molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This quenching bioluminescent voltage indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human-induced pluripotent stem cells (hiPSCs), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule-protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Diagnóstico por Imagen , Transferencia de Energía , Células HEK293 , Humanos , Luciferasas/genéticaRESUMEN
Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.
Asunto(s)
Bioensayo , Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Oxindoles/farmacología , Piperidinas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular/genética , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Células HEK293 , Humanos , Cinética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Unión Proteica , Proteolisis/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.
Asunto(s)
Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/análisis , Proteínas/análisis , Sistemas CRISPR-Cas , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , PlásmidosRESUMEN
Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi's) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi's and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi's and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi's, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteína Quinasa CDC2 , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Quinasa 9 Dependiente de la Ciclina , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Fosforilación , Relación Estructura-ActividadRESUMEN
Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).
Asunto(s)
Fibronectinas/fisiología , Malla Trabecular/fisiología , Humor Acuoso/fisiología , Fibronectinas/química , Humanos , Receptores de Fibronectina/fisiología , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Proteínas Luminiscentes/genética , Oligopéptidos/genética , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos/química , Transferencia de Energía por Resonancia de Bioluminiscencia , Proteína 9 Asociada a CRISPR/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Genes Reporteros/genética , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leupeptinas/farmacología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Luciferasas/metabolismo , Luminiscencia , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Streptococcus pyogenes/enzimologíaRESUMEN
Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.
RESUMEN
The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and "off-targets" exhibiting a range of binding affinities, cellular abundances, and binding characteristics.
Asunto(s)
Alcanos/química , Paladio/química , Proteínas/química , CatálisisRESUMEN
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.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Secuencia de Aminoácidos , Arrestinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Sustancias Luminiscentes/química , Sustancias Luminiscentes/metabolismo , Mediciones Luminiscentes/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Arrestina beta 2 , beta-Arrestinas , beta-Lactamasas/metabolismoRESUMEN
PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. METHODS: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 µg/mL C3cs or with 5 µg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 µg C3rec. RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.
Asunto(s)
Actinas/metabolismo , Adenovirus Humanos/genética , Humor Acuoso/metabolismo , Complemento C3/farmacología , Vectores Genéticos/farmacología , Virus de la Inmunodeficiencia Felina/genética , Transferasas/metabolismo , Animales , Humor Acuoso/virología , Gatos , Células Cultivadas , Modelos Animales de Enfermedad , Estudios de Seguimiento , Haplorrinos , Humanos , Técnicas de Cultivo de ÓrganosRESUMEN
Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.
Asunto(s)
Colorantes Fluorescentes/química , Luciferasas de Renilla/química , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferasas de Renilla/genética , Ingeniería de ProteínasRESUMEN
PURPOSE: To determine whether cross-linked actin networks (CLANs) formed in dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells are structurally similar to those formed after ß3 integrin activation and involve αvß3 integrin signaling. METHODS: Two HTM cell strains and an αvß3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-pretreated HTM cells were plated on fibronectin with or without ß3 integrin-activating mAb AP-5. Immunofluorescence microscopy was used to identify phalloidin-labeled CLANs and to ascertain the presence of α-actinin, PIP(2), and syndecan-4 within them. ß3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. αvß3 Integrin expression levels and the ß3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy. RESULTS: CLANs associated with either DEX treatment or ß3 integrin activation contained syndecan-4, PIP(2), and α-actinin. In the absence of mAb AP-5, LY294002 did not affect DEX-associated CLAN formation, whereas NSC23766 decreased the percentage of CLAN-positive cells by 80%. In the presence of mAb AP-5, both inhibitors decreased DEX-associated CLAN formation. DEX pretreatment increased ß3 integrin-induced CLAN formation nearly sixfold and the level of αvß3 integrin expression and activation threefold compared with control cells. Activated ß3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. αvß3 Integrin overexpression in TM-1 cells increased CLAN formation twofold. CONCLUSIONS: DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of αvß3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced ß3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism.
Asunto(s)
Actinas/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Dexametasona/farmacología , Glucocorticoides/farmacología , Integrina alfaVbeta3/metabolismo , Transducción de Señal , Malla Trabecular/efectos de los fármacos , Actinina/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Microscopía Fluorescente , Fosfatidilinositol 4,5-Difosfato/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Plásmidos , Sindecano-4/metabolismo , Malla Trabecular/metabolismo , Transfección , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
PURPOSE: To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. METHODS: Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. RESULTS: HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. CONCLUSIONS: Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
Asunto(s)
Actinas/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Vitronectina/metabolismo , Malla Trabecular/metabolismo , Adulto , Antígeno CD47/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Donantes de Tejidos , Regulación hacia Arriba , Familia-src Quinasas/fisiologíaRESUMEN
To clarify the molecular mechanism for analog potency and selectivity, we investigated the ability of 1,25(OH)(2)D(3) analogs to recruit coactivators to the vitamin D receptor (VDR) transcriptional complex. Using a modified version of the avidin-biotin complex DNA binding assay, we discovered that 20S-analogs enhance the binding of specific coactivators to the transcriptional complex relative to natural hormone and that the enhanced binding occurs independently of vitamin D response element and cell type. With the exception of two of these coactivators, DRIP205 and DRIP240, all proteins were recruited to the transcriptional complex in a dose-dependent manner. While the results do not provide an explanation for tissue selectivity of 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), they provide evidence that in the presence of a full-length side chain, the 20S configuration improves binding of specific proteins to the VDR transcriptional complex while modifications at carbon 2 do not.