RESUMEN
Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken ß-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.
Asunto(s)
Blastocisto , Edición Génica , Animales , Dependovirus/genética , Epitelio , Trompas Uterinas , Femenino , Edición Génica/métodos , Vectores Genéticos/genética , Humanos , Ratones , Oviductos/metabolismo , EmbarazoRESUMEN
K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.
Asunto(s)
Compuestos de Boro/química , Membrana Celular/metabolismo , Éteres Corona/química , Colorantes Fluorescentes/química , Potasio/metabolismo , Cationes Monovalentes , Células HEK293 , Células HT29 , Células HeLa , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Ionóforos de Potasio/farmacologíaRESUMEN
We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.
Asunto(s)
Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Proteínas Fluorescentes Verdes/química , Níquel/química , Ácido Nitrilotriacético/química , Oligopéptidos/química , Péptidos/química , Proteínas/química , Sitios de Unión , Fluorescencia , Estructura Molecular , Compuestos Organometálicos/químicaRESUMEN
The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Quinasa I-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/química , Línea Celular , Evaluación Preclínica de Medicamentos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Inmunoprecipitación , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Factores de TiempoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder. Despite its severity, there are no effective treatments because of the complexity of its pathogenesis. As one of the underlying mechanisms of Cu, Zn superoxide dismutase (SOD1) gene mutation-induced ALS, SOD1 mutants (SOD1mut) commonly interact with an endoplasmic reticulum-resident membrane protein Derlin-1, triggering motoneuron death. However, the importance of SOD1-Derlin-1 interaction in in vitro human model and in vivo mouse model remains to be elucidated. Here, we identify small-molecular-weight compounds that inhibit the SOD1-Derlin-1 interaction by screening approximately 160,000 compounds. The inhibitor prevents 122 types of SOD1mut from interacting with Derlin-1, and significantly ameliorates the ALS pathology both in motoneurons derived from patient induced pluripotent stem cells and in model mice. Our data suggest that the SOD1-Derlin-1 interaction contributes to the pathogenesis of ALS and is a promising drug target for ALS treatment.
Asunto(s)
Esclerosis Amiotrófica Lateral/prevención & control , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión Proteica/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
BACKGROUND: Prostacyclin (PGI2) is a short-lived endogenous inhibitor of platelet aggregation and a potent vasodilator and regulator of the growth of vascular smooth muscle cells. To study the role of PGI2 in the vascular system in vivo, PGI2-deficient (PGID) mice were established by genetic disruption of the PGI2 synthase gene. METHODS AND RESULTS: PGI2 synthase-null mice were generated by replacing the exons of PGI2 synthase gene that encodes for the catalytic site of the enzyme with a neomycin resistance gene. In these mice, PGI2 levels in the plasma, kidneys, and lungs were reduced, whereas thromboxane and prostaglandin E2 levels became elevated. Blood pressure and the amounts of urea nitrogen and creatinine in plasma of the PGID mice were significantly higher than those of wild-type mice (P<0.05). They developed progressive morphological abnormalities in the kidneys, accompanied by atrophy, surface irregularity, fibrosis, cyst, arterial sclerosis, and hypertrophy of vessel walls. Thickening of the thoracic aortic media and adventitia were observed in aged PGID mice. Importantly, these phenotypes have not been reported in PGI2 receptor-deficient mice. CONCLUSIONS: PGI2 deficiency resulted in the development of vascular disorders with the thickening of vascular walls and interstitial fibrosis, especially in mouse kidneys. The findings demonstrated in vivo that PGI2 is important in the homeostasis of blood vessels. Our established PGID mice are useful for studies on the initiation and development of vascular diseases, such as ischemic renal disorders with arterial sclerosis and infarction, and also for studies on the novel signaling pathway of PGI2.
Asunto(s)
Epoprostenol/deficiencia , Infarto/patología , Isquemia/patología , Riñón/patología , Nefroesclerosis/patología , Factores de Edad , Animales , Aorta Torácica/patología , Presión Sanguínea/genética , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Dinoprostona/metabolismo , Progresión de la Enfermedad , Epoprostenol/genética , Frecuencia Cardíaca/genética , Infarto/genética , Infarto/metabolismo , Oxidorreductasas Intramoleculares/deficiencia , Oxidorreductasas Intramoleculares/genética , Isquemia/genética , Isquemia/metabolismo , Riñón/irrigación sanguínea , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Nefroesclerosis/genética , Nefroesclerosis/metabolismo , Prostaglandinas/metabolismo , Tromboxano B2/metabolismoRESUMEN
An association of gallbladder carcinoma with cholelithiasis suggests that chronic inflammation may modulate tumorigenesis and/or progression of the carcinoma. An enhanced expression of cyclooxygenase-2 (COX-2) is observed frequently in advanced carcinomas of gastrointestinal tracts, which in turn suggests that potentiated arachidonate metabolism may play a crucial role in tumor biology. In the present study, the expression levels of COX-2 and prostaglandin E receptor subtypes were determined in 16 cases of gallbladder carcinomas of different depths of invasion (pT(1) 3, pT(2) 2, pT(3) 4, and pT(4) 7) to determine the role of arachidonate metabolism in tumor growth and progression. The mRNA levels of COX-2 were increased significantly in pT(3) and pT(4) carcinomas compared with the levels in pT(1) and pT(2) carcinomas. Immunohistochemistry and in situ hybridization revealed the existence of COX-2 mRNA and protein in both the cancerous epithelia and adjacent stroma of pT(1)-pT(4) carcinomas. Only in pT(3) and pT(4) carcinomas was intense expression of COX-2 observed in the adjacent stroma. The tissue concentration of PGE(2) was significantly increased in pT(3) and pT(4) carcinomas. The mRNAs of PGE receptor subtypes EP(2), EP(3), and EP(4) were amplified in pT(1)-pT(4) gallbladder carcinomas, in which their mRNAs and EP(4) protein were expressed mostly in the cancerous epithelia. Treatment with a specific EP(4) agonist, as well as PGE(2) but not EP(2) and EP(3) agonists, up-regulated the expression of c-fos, an induced growth response gene, and increased colony formation. In advanced gallbladder carcinoma, enhanced expression of COX-2 is observed in the adjacent stroma rather than in the cancerous epithelia, and the stroma is a potent source of PG synthesis. In epithelial-stromal interactions, the increased PGE(2) synthesis in the adjacent stroma and its biological effect via EP(4) on the carcinoma cells may contribute to tumor growth and progression of gallbladder carcinoma.
Asunto(s)
Neoplasias de la Vesícula Biliar/patología , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores de Prostaglandina E/metabolismo , Anciano , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , División Celular/efectos de los fármacos , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Progresión de la Enfermedad , Femenino , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/metabolismo , Fosfolipasas A2 Grupo II , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Prostaglandina E/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.
Asunto(s)
Gliotoxina/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Inmunosupresores/farmacología , FN-kappa B/antagonistas & inhibidores , Ubiquitina/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Fluoresceína/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , Gliotoxina/química , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunosupresores/química , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de Señal , Terbio/química , Factores de Transcripción , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina/genética , Ubiquitina/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitinación/efectos de los fármacos , Ubiquitinas/genética , Ubiquitinas/inmunologíaRESUMEN
Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Oxidorreductasas Intramoleculares/química , Proteínas Recombinantes/química , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/química , 6-Cetoprostaglandina F1 alfa/química , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Baculoviridae/genética , Línea Celular , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Durapatita/química , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/aislamiento & purificación , Cinética , Oxidación-Reducción , Mutación Puntual , Endoperóxidos de Prostaglandinas Sintéticos/química , Prostaglandina H2/química , Prostaglandina H2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría , Spodoptera , Tromboxano A2/análogos & derivados , Transfección , Tranilcipromina/químicaRESUMEN
Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.