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Bioluminescence and kinetic aspects of double mutated aequorin variants.

Aequorin as an old small calcium-sensitive photoprotein is a blue fluorescence protein which converts coelenterazine (a substrate) to coelenteramide with a flash type emission. The decay kinetics and emission properties of this protein can be changed using directed mutagenesis of crucial amino acid residue. In this work, we prepared three double mutants: YF/WF, YF/DG, and WF/DG. According to our results, it seems that presence of YF mutation results in shift of emission to longer wavelengths while the WF mutation shifts the emission to shorter wavelengths. Furthermore, comparison of the variants for light half-life indicated decreased tfor the two variants of YF/DG and WF/DG. But in compared to wild type aequorin, the YF/WF variant displayed a 2-fold increase of light half-life. On the other hand, the thermostability properties of double mutants confirmed that only YF/DG variant of apoaequorin is higher stability than others. Also, the single WF mutant reached the highest stability against thermal shock. Our data suggest that replacement of single or few point mutations in the binding pocket or active site of aequorin affects its bioluminescence and kinetic properties and so could be used for new reporter production of this photoprotein with the feasibility and limited substitutions.

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Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

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Semisynthetic photoprotein reporters for tracking fast Ca(2+) transients.

Changes in the intracellular concentration of free ionized calcium ([Ca(2+)]i) control a host of cellular processes as varied as vision, muscle contraction, neuronal signal transmission, proliferation, apoptosis etc. The disturbance in Ca(2+)-signaling causes many severe diseases. To understand the mechanisms underlying the control by calcium and how disorder of this regulation relates to pathological conditions, it is necessary to measure [Ca(2+)]i. The Ca(2+)-regulated photoproteins which are responsible for bioluminescence of marine coelenterates have been successfully used for this purpose over the years. Here we report the results on comparative characterization of bioluminescence properties of aequorin from Aequorea victoria, obelin from Obelia longissima, and clytin from Clytia gregaria charged by native coelenterazine and coelenterazine analogues f, i, and hcp. The comparison of specific bioluminescence activity, stability, emission spectra, stopped-flow kinetics, sensitivity to calcium, and effect of physiological concentrations of Mg(2+) establishes obelin-hcp as an excellent semisynthetic photoprotein to keep track of fast intracellular Ca(2+) transients. The rate of rise of its light signal on a sudden change of [Ca(2+)] is almost 3- and 11-fold higher than those of obelin and aequorin with native coelenterazine, respectively, and 20 times higher than that of the corresponding aequorin-hcp. In addition, obelin-hcp preserves a high specific bioluminescence activity and displays higher Ca(2+)-sensitivity as compared to obelin charged by native coelenterazine and sensitivity to Ca(2+) comparable with those of aequorin-f and aequorin-hcp.

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Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.

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C6-Deoxy coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanoKAZ: the mutated catalytic 19 kDa component of Oplophorus luciferase.

The codon-optimized gene for the mutated 19 kDa protein (nanoKAZ), which is the catalytic component of Oplophorus luciferase, was expressed in Escherichia coli cells and the recombinant protein was highly purified. The secretory expression of nanoKAZ from CHO-K1 cells was performed by fusing the secretory signal peptide sequence of Gaussia luciferase to the amino-terminus of nanoKAZ. The substrate specificity for the purified nanoKAZ and the nanoKAZ secreted into the cultured medium was determined, indicating that bis-coelenterazine (bis-CTZ) and newly synthesized 6h-f-coelenterazine (6h-f-CTZ) are an efficient substrate for the glow luminescence reaction of nanoKAZ.

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Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.

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Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans.

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid", Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189-197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg2+, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6-2 microm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 degrees C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-gamma-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.

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Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals.

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

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Bioluminescence of Aequorea macrodactyla, a common jellyfish species in the East China Sea.

Studies of the bioluminescent mechanisms of jellyfish have been mainly confined to one species, Aequorea victoria. We describe the luminescent system of another species, Aequorea macrodactyla, which is commonly found in the warmer waters on the coastal region of East China Sea. The luminescent system of this species consists of a green fluorescent protein (GFP) and one or more aequorins. The GFP gene is 1042 bp. It encompasses a coding sequence of 717 bp organized as 3 exons, and it is predicted to specify a 27-kDa peptide, which shares 80% amino acid sequence identity with the GFP of A. victoria. The entire coding sequence was cloned into the pTO-T7 expression vector and expressed in Escherichia coli. Compared with GFP of A. victoria, the purified expressed protein exhibited an excitation peak at a higher wavelength of 476 nm and an emission peak at a lower wavelength of 496 nm, with a higher quantum yield of 1.0. The other photoprotein, aequorin, is encoded in a single open reading frame of 585 bp specifying a 23-kDa apoprotein. The gene was cloned in to the same expression vector and expressed in E. coli. The activity of the photoprotein was reconstituted by incubating the expressed apoprotein with coelenterazine f. In the presence of Ca(2+) the reconstituted aequorin exhibits an emission peak at 470 nm. The kinetics of regeneration and the photoactivities of the reconstituted aequorins of the 2 species of jellyfish are similar. Nevertheless, Aequorea macrodactyla is expected to appear brighter and more "blue" than Aequorea victorea because of the differences in the photoactivity of their GFPs.

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