RESUMO
Bioluminescence is the emission of visible light by living organisms. Here we describe the isolation and characterisation of a cDNA encoding a MW ≈ 59,000 Da luciferase from the Australian glow-worm, Arachnocampa richardsae. The enzyme is a member of the acyl-CoA ligase superfamily and produces blue light on addition of D-luciferin. These results are contrary to earlier reports (Lee, J., Photochem Photobiol 24, 279-285 (1976), Viviani, V. R., Hastings, J. W. & Wilson, T., Photochem Photobiol 75, 22-27 (2002)), which suggested glow-worm luciferase has MW ≈ 36,000 Da and is unreactive with beetle luciferin. There are more than 2000 species of firefly, which all produce emissions from D-luciferin in the green to red regions of the electromagnetic spectrum. Although blue-emitting luciferases are known from marine organisms, they belong to different structural families and use a different substrate. The observation of blue emission from a D-luciferin-using enzyme is therefore unprecedented.
Assuntos
Benzotiazóis/metabolismo , Dípteros/metabolismo , Proteínas de Insetos/metabolismo , Luciferases/metabolismo , Animais , Austrália , DNA Complementar/genética , Dípteros/química , Dípteros/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Luciferases/química , Luciferases/genética , Luminescência , Medições Luminescentes , Especificidade por SubstratoRESUMO
Altered tissue water homeostasis may contribute to edema formation during various stresses including bacterial infection. We observed induction of aquaporin-1 (AQP1) during Staphylococcus aureus infection of cultured cells indicating a potential mechanism underlying altered water homeostasis during infection. To investigate mechanisms of AQP1 induction, we examined the effects of the S. aureus alpha-hemolysin on AQP1 abundance in Balb/c fibroblasts. Fibroblasts incubated with 30 microg/ml hemolysin exhibited a 5-10 fold increase in AQP1 protein within 4-6 hours of exposure. The use of multiple signaling cascade inhibitors failed to affect hemolysin-mediated accumulation of AQP1. However, immunoprecipitation revealed an initial accumulation of ubiquitinated AQP1 followed by a decrease to baseline levels after 4 hours. Immunofluorescence indicated that following hemolysin exposure, AQP1 was no longer on the plasma membrane, but was found in a population of submembrane vacuoles. AQP1 redistribution was further indicated by surface biotinylation experiments suggesting diminished AQP1 abundance on the plasma membrane as well as redistribution out of lipid raft fractions. Live cell confocal microscopy revealed that the pattern of cell volume change observed following hemolysin exposure was altered in cells in which AQP1 was silenced. We conclude that alpha-toxin alters proteasomal processing and leads to intracellular accumulation of AQP1, which may likely contribute to disrupted cell volume homeostasis in infection.
Assuntos
Aquaporina 1/metabolismo , Toxinas Bacterianas/farmacologia , Proteínas Hemolisinas/farmacologia , Necrose/patologia , Animais , Aquaporina 1/genética , Tamanho Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteassoma , Staphylococcus aureus , Ubiquitinação/efeitos dos fármacosRESUMO
Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Mammalian cells are confronted with changes in extracellular osmolality at various sites, including the aqueous layer above the lung epithelium. Hypertonic shock induces the activation of mitogen-activated protein kinases and the expression of a defined set of genes, including aquaporins. We investigated upstream components of the response to hypertonicity in lung epithelial cells and found that before extracellular signal-regulated kinase activation and aquaporin synthesis, the membrane-bound prohormone neuregulin 1-beta is cleaved and binds to human epidermal growth factor receptor 3 (HER3). The signaling is prevented by matrix metalloproteinase inhibition, inhibition of neuregulin 1-beta binding to HER3, and inhibition of HER tyrosine kinase activity. Inhibition of HER activation interferes with the hypertonic induction of two different aquaporins in three distinct cell lines of mouse and human origin. We propose that ligand-dependent HER activation constitutes a generalized signaling principle in the mammalian hypertonic stress response relevant to aquaporin expression.