RESUMEN
Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.
Asunto(s)
Genes Supresores de Tumor , Proteínas Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Neoplasias/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-beta-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca(2+) and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Galphai G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Galphai G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Galphai G-proteins.
Asunto(s)
Endocitosis , Microdominios de Membrana/metabolismo , Receptores CCR5/metabolismo , Transducción de Señal , Animales , Células CHO , Calcio/metabolismo , Caveolina 1/metabolismo , Quimiocina CCL3/farmacología , Colforsina/farmacología , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Endocitosis/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Toxina del Pertussis/farmacología , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
SCOPE: Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti-inflammatory effects of flavonoid metabolites relative to their precursor structures. METHODS AND RESULTS: Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1-10 µM) were screened for their ability to reduce LPS-induced tumor necrosis factor-α (TNF-α) secretion in THP-1 monocytes. One micromolar peonidin-3-glucoside, cyanidin-3-glucoside, and the metabolites isovanillic acid (IVA), IVA-glucuronide, vanillic acid-glucuronide, protocatechuic acid-3-sulfate, and benzoic acid-sulfate significantly reduced TNF-α secretion when in isolation, while there was no effect on TNF-α mRNA expression. Four combinations of metabolites that included 4-hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF-α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS-induced IL-1ß and IL-10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL-1ß secretion but none of the flavonoids or metabolites significantly modified IL-10 secretion. CONCLUSION: This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites.
Asunto(s)
Flavonoides/farmacología , Monocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Flavonoides/química , Humanos , Hidroxibenzoatos/farmacología , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/efectos adversos , FN-kappa B/genética , FN-kappa B/metabolismo , Parabenos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
CC and CXC chemokine receptor signalling networks are regulated in different ways. Here we show that intracellular calcium release and cell migration occur independent of Gßγ activation in response to CCL3, whereas CXCL11 induced migration of activated T-lymphocytes depends on Gßγ activation. Treatment of a range of cell types with gallein, a pharmacological inhibitor of Gßγ signalling, did not result in a reduction in CCL3 induced cellular migration, but resulted in enhanced calcium mobilisation following chemokine stimulation. Inhibition of PI3 kinase (PI3K) and AKT, which are activated downstream of Gßγ, equally had no effect on calcium release and a minor effect on cell migration. Similarly, inhibition of ERK1/2 did not prevent CCL3 induced migration. Interestingly, Gßγ as well as PI3K activation is necessary for CXCL11 induced migration of activated T-cells. These data not only confirm a role for Gßγ signalling in CXCL11 induced migration, but also demonstrate that targeting Gßγ as a therapeutic target to prevent migration in inflammatory disease may not be beneficial, at least not for CCL3 induced migration. This highlights the distinct differences in the mechanisms on how CC- and CXC-receptors activate cellular migration.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Células CHO , Calcio/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocina CCL3/farmacología , Quimiocina CXCL11/farmacología , Quimiotaxis/efectos de los fármacos , Cricetinae , Cricetulus , Células HEK293 , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CCR5/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Xantenos/farmacologíaRESUMEN
Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods. Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t(1/2) ≤ 0.1 sec) with a low false positive rate. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters. The recombinant protein libraries are produced using a convenient and high-level mammalian expression system, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (ß-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates.