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Find video protocols related to scientific articles indexed in Pubmed.
Chemokine cooperativity is caused by competitive glycosaminoglycan binding.
J. Immunol.
PUBLISHED: 03-17-2014
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Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.
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GPCR-targeting nanobodies: attractive research tools, diagnostics, and therapeutics.
Trends Pharmacol. Sci.
PUBLISHED: 01-10-2014
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G-protein-coupled receptors (GPCRs) represent a major therapeutic target class. A large proportion of marketed drugs exert their effect through modulation of GPCR function, and GPCRs have been successfully targeted with small molecules. Yet, the number of small new molecular entities targeting GPCRs that has been approved as therapeutics in the past decade has been limited. With new and improved immunization-related technologies and advances in GPCR purification and expression techniques, antibody-based targeting of GPCRs has gained attention. The serendipitous discovery of a unique class of heavy chain antibodies (hcAbs) in the sera of camelids may provide novel GPCR-directed therapies. Antigen-binding fragments of hcAbs, also referred to as nanobodies, combine the advantages of both small molecules (e.g., molecular cavity binding, low production costs) and monoclonal antibodies (e.g., high affinity and specificity). Nanobodies are gaining ground as therapeutics and are also starting to find application as diagnostics and as high-quality tools in GPCR research. Herein, we review recent advances in the use of nanobodies in GPCR research.
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?-Arrestin recruitment and G protein signaling by the atypical human chemokine decoy receptor CCX-CKR.
J. Biol. Chem.
PUBLISHED: 01-22-2013
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Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with ?-arrestin2-GFP translocation. Moreover, recruitment of ?-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold ?-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.
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Discovery of novel small molecule activators of ?-catenin signaling.
PLoS ONE
PUBLISHED: 03-22-2011
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Wnt/?-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/?-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a ?-galactosidase fragment complementation assay measuring ?-catenin nuclear entry revealed bona fide activators of ?-catenin signaling. The compounds stabilized cytoplasmic ?-catenin and activated ?-catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate ?-catenin signaling has yet to be determined, several key regulators of Wnt/?-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced ?-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/?-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/?-catenin signaling.
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Inhibition of Wnt/?-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase I?/?.
Chem. Biol.
PUBLISHED: 01-26-2011
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Wnt/?-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a ?-galactosidase fragment complementation assay measuring ?-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated ?-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/?-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated ?-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase I? and ?, which are known activators of Wnt/?-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of ?-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/?-catenin signaling.
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A model for signaling specificity of Wnt/Frizzled combinations through co-receptor recruitment.
FEBS Lett.
PUBLISHED: 07-16-2010
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Wnts control mammalian developmental morphogenesis and are critical for adult stem cell maintenance. Wnts initiate several intracellular signaling cascades, such as Wnt/?-catenin-, Wnt/Ca(2+)- and Wnt/ROR2-signaling. Signaling preference of Wnts for these various pathways is thought to depend on the repertoire of receptors present on recipient cells. Here, we propose a further refinement of this receptor model and hypothesize that Wnt signaling specificity depends on co-receptor recruitment upon binding of Wnt to Frizzled receptor molecules. In this model, recruitment of LRP5/6 leads to activation of Wnt/?-catenin signaling, whereas signaling through other pathways is mediated by recruiting ROR2.
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New avenues to target Wnt/?-catenin signaling.
Drug Discov. Today
PUBLISHED: 07-14-2010
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Wnt/?-catenin signaling is an evolutionarily conserved signaling cascade with imperative roles during development and in adult stem cell maintenance. Hyperactivation of Wnt/?-catenin drives various cancers, whereas hypoactivation underlies bone malformations and neurodegenerative disorders. Although several small molecule modulators of Wnt/?-catenin signaling have been identified, none have progressed into clinical trials yet. Recent studies employing genomics and proteomics approaches have yielded more druggable targets, such as kinases and seven-transmembrane receptors. In addition, new assay methods enable a more targeted approach for high-throughput screening of this pathway and are expected to deliver clinical candidates in the coming decade.
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beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling.
FASEB J.
PUBLISHED: 11-25-2009
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Wnt/beta-catenin signaling is an important regulator of cell polarity, proliferation, and stem cell maintenance during development and adulthood. Wnt proteins induce the nuclear accumulation of beta-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/beta-catenin signaling has been implicated in a plethora of pathologies and, most notably, underlies initiation and expansion of several cancers. Here, we apply enzyme fragment complementation to measure the nuclear accumulation of beta-catenin. beta-Catenin was tagged with a peptide fragment of beta-galactosidase and transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus. Stimulation of the cells with recombinant Wnt-3a restored beta-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses beta-catenin-driven reporter gene activity downstream of nuclear entry of beta-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce beta-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was identified as an activator of Wnt/beta-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous beta-catenin protein and can induce TCF/LEF-dependent gene transcription. Subsequent biochemical profiling of >200 kinases revealed both isoforms of glycogen synthase kinase 3, as previously unappreciated targets of sotrastaurin. We show that the beta-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/beta-catenin pathway and can be used to find new therapeutics targeting Wnt/beta-catenin signaling.-Verkaar, F., Blankesteijn, W. M., Smits, J. F. M., Zaman, G. J. R. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling.
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Stably overexpressed human Frizzled-2 signals through the beta-catenin pathway and does not activate Ca2+-mobilization in Human Embryonic Kidney 293 cells.
Cell. Signal.
PUBLISHED: 02-07-2009
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Signal transduction via the Frizzled family of seven-transmembrane receptors controls important developmental processes. Aberrant signaling caused by altered Frizzled receptor activity or by mutations in downstream signaling components has been implicated in several adult pathologies. A diverse array of intracellular signaling pathways has been suggested to transduce the signals exerted in cells when secreted ligands of the Wnt family bind to Frizzled receptors. Studies with a chimeric receptor composed of Frizzled-2 and the beta2-adrenergic receptor have suggested that the binding of Wnt-5a to Frizzled-2 results in the activation of G proteins of the Galpha(i) family, the mobilization of calcium from intracellular stores and the induction of gene transcription through nuclear factor of activated T cells. In this report, we demonstrate by using beta-lactamase reporter gene technology that full-length, wild-type human Frizzled-2 does not couple to calcium-mediated signaling in HEK293 cells following stimulation with purified recombinant mouse Wnt-5a. In contrast, when stimulated with recombinant mouse Wnt-3a, Frizzled-2 activates the canonical Wnt/Frizzled signaling pathway, involving the transcriptional modulator beta-catenin. Our report underlines the importance of using cell lines stably overexpressing wild-type Frizzled receptors and the use of purified ligands when studying receptor pharmacology. This approach has allowed us to measure the half-maximal concentration for activation of human Frizzled-2 (1.5+/-0.4 nM; avg.+/-SD) and human Frizzled-1 (1.3+/-0.5 nM) following stimulation by Wnt-3a. Our results suggest that there is receptor redundancy with regard to Wnt-3a reception. In addition, we introduce beta-lactamase reporter gene technology as an alternative to luciferase-based reporters to measure Frizzled receptor modulation for the discovery of Frizzled receptor-interacting drugs.
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Celebrating 30 years of Wnt signaling.
Sci Signal
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Wnt signal transduction is crucial for embryonic development and tissue homeostasis in multicellular animals. Hyperactivation of the Wnt pathway drives tumor formation, yet activation of the Wnt pathway in stem cells holds great promise for injury repair and regeneration. Between 27 June and 1 July 2012, scientists from all over the globe gathered in the beachfront town of Egmond aan Zee in the Netherlands to celebrate the 30th anniversary of this blossoming and exciting field. The latest advances and breakthroughs were discussed at the aptly named European Molecular Biology Organization conference 30 Years of Wnt Signalling. Many presenters discussed unpublished data, a hallmark of past and hopefully future Wnt meetings. This Meeting Report summarizes some of the highlights of this conference, including the presentation of the long-awaited crystal structure of a Wnt protein bound to its receptor and the identification of exciting new possibilities for targeting the pathway in treating disease.
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?-Arrestin-biased signaling of PTH analogs of the type 1 parathyroid hormone receptor.
Cell. Signal.
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Parathyroid hormone (PTH) is an anabolic agent that mediates bone formation through activation of the G?(s)-, G?(q)- and ?-arrestin-coupled parathyroid hormone receptor type 1 (PTH1R). Pharmacological evidence based on the effect of PTH(7-34), a PTH derivative that is said to preferentially activate ?-arrestin signaling through PTH1R, suggests that PTH1R-activated ?-arrestin signaling mediates anabolic effects on bone. Here, we performed a thorough evaluation of PTH(7-34) signaling behaviour using quantitative assays for ?-arrestin recruitment, G?(s)- and G?(q)-signaling. We found that PTH(7-34) inhibited PTH-induced cAMP accumulation, but was unable to induce ?-arrestin recruitment, PTH1R internalization and ERK1/2 phosphorylation in HEK293, CHO and U2OS cells. Thus, the ?-arrestin bias of PTH(7-34) is not apparent in every cell type examined, suggesting that correlating in vivo effects of PTH(7-34) to in vitro pharmacology should be done with caution.
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Constitutive ?-catenin signaling by the viral chemokine receptor US28.
PLoS ONE
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Chronic activation of Wnt/?-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ?-catenin. In this study we show that this viral receptor constitutively activates ?-catenin and enhances ?-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ?-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ?-catenin. Moreover, cells infected with HCMV show significant increases in ?-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ?-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases.
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A prospective cross-screening study on G-protein-coupled receptors: lessons learned in virtual compound library design.
J. Med. Chem.
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We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the ?-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.
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A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets.
Br. J. Pharmacol.
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To establish the druggability of a target, genetic validation needs to be supplemented with pharmacological validation. Pharmacological studies, especially in the kinase field, are hampered by the fact that many reference inhibitors are not fully selective for one target. Fortunately, the initial trickle of selective inhibitors released in the public domain has steadily swelled into a stream. However, rationally picking the most selective tool compound out of the increasing amounts of available inhibitors has become progressively difficult due to the lack of accurate quantitative descriptors of drug selectivity. A recently published approach, termed selectivity entropy, is an improved way of expressing selectivity as a single-value parameter and enables rank ordering of inhibitors. We provide a guide to select the best tool compounds for pharmacological validation experiments of candidate drug targets using selectivity entropy. In addition, we recommend which inhibitors to use for studying the biology of the 20 most investigated kinases that are clinically relevant: Abl (ABL1), AKT1, ALK, Aurora A/B, CDKs, MET, CSF1R (FMS), EGFR, FLT3, ERBB2 (HER2), IKBKB (IKK2), JAK2/3, JNK1/2/3 (MAPK8/9/10), MEK1/2, PLK1, PI3Ks, p38? (MAPK14), BRAF, SRC and VEGFR2 (KDR).
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.