We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.
In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.
Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca 2ϩ accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca 2ϩ channel inhibition and K ϩ channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K ϩ current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-␣, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K ϩ channel activation and protection against ischemia-reperfusion injury.
For spatial and quantitative kinetic analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence using a cell membrane permeable substrate, coelenterazine. This split Renilla luciferase complementation readout was shown to work for locating a PPI between the tyrosine-phosphorylated peptide (Y941) of IRS-1 and the SH2 domain of PI3K among insulin signaling pathways in living Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR). It was thereby found that the insulin-stimulated interaction occurred near the plasma membrane in the cytosol.
For spatial and quantitative analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed, in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence, with a cell membrane permeable substrate, colenterazine. Consequently, unlike conventional complement enzymes that lead to stable diffusive fluorescent products, this split Renilla luciferase complementation readout is capable of locating the PPIs with emission of bioluminescence only at the sites and time of their occurrence in living cells. To monitor the interaction between two proteins A and B, N-terminal half of the split Renilla luciferase is fused to protein A, and protein B to C-terminal half of the split Renilla luciferase. Interaction between protein A and protein B, and the consequent juxtapositioning of the split Renilla luciferase simultaneously leads to formation of the complement Renilla luciferase, thereby spontaneously emitting bioluminescence with its cell membrane permeable substrate, coelenterazine in situ in living mammalian cells. This split Renilla luciferase complementation readout was shown to work for locating a PPI between tyrosine-phosphorylated peptide (Y941) of IRS-1 and SH2 domain of PI3K among insulin signaling pathways in living Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR). It was thereby found that the insulin stimulated interaction occurred near to the plasma membrane in the cytosol. The PPI between Shc and Grb2 was also studied with the same technique.
Abstract. We recently reported that physiological concentrations of 17β-estradiol partially down-regulate cardiac rapidly-activating delayed rectifier K + currents (hERG currents) independently of estrogen-receptor signaling. To determine if other estrogens have the same effect as that of 17β-estradiol, we investigated receptor-independent effects of estrone, estrone 3-sulfate, and estriol on hERG currents in patch-clamped estrogen-negative HEK293 cells. Only estrone 3-sulfate partially suppressed hERG currents in a receptor-independent manner by modifying the gating. The concentration-dependence of estrone 3-sulfate revealed that physiological levels of circulating estrone 3-sulfate can modulate hERG currents to the maximal extent in both women and men at any age.Female gender is an independent risk factor for development of torsade de pointes (TdP) not only in congenital long QT syndromes (LQTS) but also in acquired LQTS (1, 2) resulting mostly from blockade of the human ether-a-go-go-related gene (hERG) channel (3). Actually 65% -75% of drug-induced TdP occurs in women, and is thought to be associated, at least in part, with longer baseline rate-corrected QT intervals (QT C ) in women than those in men (4).There is increasing evidence that effects of sex hormones on the ionic process underlying cardiac repolarization are important determinants of the sexrelated differences (5 -7). The shortening of the QT interval by testosterone and / or progesterone have been suggested from clinical reports and model studies. In contrast, some clinical studies imply that estrogen can increase the risks of drug-induced TdP in women. In healthy premenopausal women volunteers, the susceptibility of drug-induced QT C prolongation is exaggerated in the late follicular phase where estrogen level is the highest (8). Baseline QT C in postmenopausal women who are currently taking an only estrogen-replacement therapy is slightly but significantly longer compared with the control population (9). In addition to the well-characterized influences of estrogen in cardiac repolarization concerning hormonal genomic actions (1, 10), we recently found that the physiological levels of 17β-estradiol (E2), the bioactive estrogen, acutely downregulates the hERG channel, resulting in QT C prolongation (11). Such effects of E2 are independent of the estrogen-receptor signaling, and interaction between aromatic rings of E2 and F656 hERG may be important for the effect (11).Despite of these intensive studies, underlying mechanisms for a contribution of estrogen to arrhythmogenicity remains to be unclear: for example, It has been shown that propensity to drug-induced TdP does not decline after menopause in women who have very low levels of endogenous ovarian estrogen including E2 (12). Because the hERG suppression by E2 is independent of the estrogen-receptor signaling (11), it is possible that other estrogens suppress hERG currents as E2 does. However there have been no reports as to the ability of other estrogens to modulate hERG currents acutely.To ...
Moreover, chimera analysis reveals a region of KCNE1 sufficient to confer cAMP-dependent functional regulation upon the KCNQ1_ KCNE3_Yotiao channel. The property of specific beta subunits to transduce post-translational regulation of alpha subunits of ion channels adds another dimension to our understanding molecular mechanisms underlying the diversity of regulation of native K + channels.
In this study, a genetically encoded bioluminescent indicator for ERK2 dimer was developed with the split Renilla luciferase complementation method, in which the formation of ERK2 dimer induces a spontaneous emission of bioluminescence in living cells. In response to extracellular stimuli, such as epidermal growth factor (EGF) or 17beta-estradiol (E2), extracellular signal-regulated kinase 2 (ERK2) is phosphorylated by its upstream kinase MEK, and also phosphorylates its substrates in various regions of the cell, including the nucleus. Phosphorylated ERK2 is led to form its dimer, thereby transporting itself into the nucleus. We demonstrated with the indicator that stimulation with EGF or E2 induces the formation of ERK2 dimer in living MCF-7 cells. The dynamics of this dimer formation was examined and discussed.
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