[show abstract][hide abstract] ABSTRACT: Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.
Proceedings of the National Academy of Sciences 09/2004; 101(32):11542-7. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo.
[show abstract][hide abstract] ABSTRACT: Determination of protein cytokines in local tissues would help to evaluate their local role in health, sickness behavior and immune-mediated diseases. Therefore, developing a simple quantitative method of protein cytokines in tissues/organs is highly important.
Mouse tissues were collected following intraperitoneal administration of endotoxin-free PBS or lipopolysaccharide. A mild detergent, 0.1% Igepal, was added in a buffer to enhance cytokines extraction. The tissues were then disrupted, homogenized, centrifuged and the supernatants were collected and assayed using solid-phase immunoassays.
The presence of 0.1% Igepal extracted significantly more TNF-alpha from liver (322%: p<0.01), brain (358%: p<0.05), lungs (1600%: p<0.01), and more IL-10 from liver (220%: p<0.001), brain (4650%: p<0.001) than PBS alone. On the other hand, using 0.1% Igepal did not increase IFN-gamma extraction from liver, spleen, brain, lungs, skin and kidneys more than PBS alone. Furthermore, i.p. administration of LPS induced a differential milieu of cytokines. LPS increased significantly the production of TNF-alpha, IFN-gamma, and IL-10 from liver (521%, 123%, 72%: p<0.01, 0.04, 0.04), brain (470%, 122%, 280%: p< 0.01, 0.03, 0.01), peritoneal lavage (p<0.001) and blood (p<0.001). However, the pattern of increase was different for the above cytokines in spleen, skin, lungs and kidneys.
The extraction of protein cytokines from tissues was superior with addition of mild detergent. Furthermore, our results showed a differential cytokines response to LPS with respect to tissue and cytokine type. This method should provide an important tool for studying local protein cytokines in behavioral pattern, sickness behavior, and immune-mediated diseases as well as to determine local therapeutic efficacy of immunomodulatory drugs.
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