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      A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines

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          Abstract

          We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147–149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542.

          The summary of the method is:

          • Cytokine activities are determined within 2 h after stimulation.

          • Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.

          • Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity.

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          Most cited references10

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          Anti-TNF-alpha therapies: the next generation.

          The functioning of the immune system is finely balanced by the activities of pro-inflammatory and anti-inflammatory mediators or cytokines. Unregulated activities of these mediators can lead to the development of serious inflammatory diseases. In particular, enhanced tumour-necrosis factor-alpha (TNF-alpha) synthesis is associated with the development of rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. Inhibiting TNF-alpha activities in these diseases has been remarkably successful. However, the current injectable protein therapies have associated risks and limitations. An oral, small molecule that regulates TNF-alpha biology could either replace the injectables or provide better disease control when used alone or in conjunction with existing therapies. In this review, we discuss briefly the present understanding of TNF-alpha-mediated biology and the current injectable therapies in clinical use, and focus on some of the new therapeutic approaches with oral, small-molecule inhibitors.
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            Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

            In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.
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              Real-time imaging of ligand-induced IKK activation in intact cells and in living mice.

              The transcription factor NF-kappaB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-kappaB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IkappaB alpha degradation by the 26S proteasome is a critical NF-kappaB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IkappaB alpha-firefly luciferase (IkappaB alpha-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IkappaB alpha-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-kappaB transcriptional reporters for more complete temporal and regional investigations of the NF-kappaB signaling pathway in health and disease.
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                Author and article information

                Contributors
                Journal
                MethodsX
                MethodsX
                MethodsX
                Elsevier
                2215-0161
                07 July 2016
                2016
                07 July 2016
                : 3
                : 483-489
                Affiliations
                [a ]Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan
                [b ]Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan
                Author notes
                [* ]Corresponding author. umezawa@ 123456chem.s.u-tokyo.ac.jp
                Article
                S2215-0161(16)30025-5
                10.1016/j.mex.2016.06.001
                4961787
                27489781
                935fb84d-c188-4e69-91a3-4b64db01127e
                © 2016 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 3 October 2015
                : 23 June 2016
                Categories
                Chemistry

                genetically encoded bioluminescent indicator based on protein splicing,bioluminescence,protein splicing assay (psa),luciferase,cytokine,nuclear factor-κb (nf-κb),tumor necrosis factor-α (tnf-α),nuclear trafficking

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