Coelenterazine sulfotransferase from Renilla muelleri.

The luciferin sulfokinase (coelenterazine sulfotransferase) of Renilla was previously reported to activate the storage form, luciferyl sulfate (coelenterazine sulfate) to luciferin (coelenterazine), the substrate for the luciferase bioluminescence reaction. The gene coding for the coelenterazine sulfotransferase has not been identified. Here we used a combined proteomic/transcriptomic approach to identify and clone the sulfotransferase cDNA. Multiple isoforms of coelenterazine sulfotransferase were identified from the anthozoan Renilla muelleri by intersecting its transcriptome with the LC-MS/MS derived peptide sequences of coelenterazine sulfotransferase purified from Renilla. Two of the isoforms were expressed in E. coli, purified, and partially characterized. The encoded enzymes display sulfotransferase activity that is comparable to that of the native sulfotransferase isolated from Renilla reniformis that was reported in 1970. The bioluminescent assay for sensitive detection of 3'-phosphoadenosine 5'-phosphate (PAP) using the recombinant sulfotransferase is demonstrated.

Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase.

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.

A hybrid de novo assembly of the sea pansy (Renilla muelleri) genome.

BACKGROUND: More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral "forests," which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals. Here, we present a de novo assembly of an azooxanthellate shallow-water octocoral, Renilla muelleri. FINDINGS: We generated a hybrid de novo assembly using MaSuRCA v.3.2.6. The final assembly included 4,825 scaffolds and a haploid genome size of 172 megabases (Mb). A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome may be smaller (172 Mb minimum size) than other publicly available coral genomes (256-448 Mb), the R. muelleri genome is similar to other coral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models. CONCLUSIONS: The R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity.

In trans promoter activation by enhancers in transient transfection.

Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

Super RLuc8: A novel engineered Renilla luciferase with a red-shifted spectrum and stable light emission.

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value=0.0084) increase in the thermostability at 37°C after 20min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37°C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

Quenching the firefly bioluminescence by various ions.

The luciferase reporter gene assay system is broadly applied in various biomedical aspects, including signaling pathway dissection, transcriptional activity analysis, and genetic toxicity testing. It significantly improves the experimental accuracy and reduces the experimental error by the addition of an internal control. In the current research, we discovered some specific ions that could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase in vitro in the dual-reporter gene assay. We showed that these ionic compounds had a high potential of being utilized as quench-and-activate reagents in the dual-reporter assay. Furthermore, results from kinetic studies on ion-mediated quenching effects indicated that different ions have distinct inhibition modes. Our study is anticipated to guide a more affordable design of quench-and-activate reagents in biomedicine and pharmaceutical analysis.

Delivery of anti-microRNA-21 antisense-oligodeoxynucleotide using amphiphilic peptides for glioblastoma gene therapy.

Inhibition of microRNA-21 (miR-21) has been shown to promote apoptosis of cancer cells and to reduce tumor size in glioblastoma. However, efficient carriers for antisense-oligodeoxynucleotide (antisense-ODN) against miR-21 have not yet been developed. In this study, the R3V6 peptide (R3V6) was evaluated as a carrier of antisense-ODN. In a gel retardation assay, R3V6 formed a complex with an antisense-ODN. The serum stability assay showed that R3V6 protected it from nucleases more efficiently than polyethylenimine (PEI; 25 kDa, PEI25k). A Renilla luciferase gene with a 3'-untranslated region (3'-UTR) recognizable by miR-21 (psiCHECK2-miR-21-UTR) was constructed for the antisense-ODN assay. psiCHECK2-miR-21-UTR expressed less Renilla luciferase in the cells with a higher level of miR-21 due to the effect of miR-21. In an in vitro transfection assay, the R3V6 peptide delivered anti-miR-21 antisense-ODN into cells more efficiently than PEI (25 kDa, PEI25k) and lipofectamine. As a result, antisense-ODN/R3V6 complex inhibited miR-21 and increased Renilla luciferase expression more efficiently than antisense-ODN/PEI25k or antisense-ODN/Lipofectamine complexes in both C6 and A172 glioblastoma cells. Furthermore, the antisense-ODN/R3V6 complexes reduced the level of miR-21 and induced apoptosis of glioblastoma cells. These results suggest that the R3V6 peptide may be a useful carrier of antisense-ODN for glioblastoma gene therapy.

Novel functional Renilla luciferase mutant provides long-term serum stability and high luminescence activity.

Fluorescent and luminescent chemical probes are essential in recent medical diagnostics. However, the use of these probes in vivo has raised concerns due to their low sensitivity, background signal interference, and non-biocompatibility. Therefore, biological chromophores have received much attention as new alternatives. In particular, luciferase, a class of oxidative enzyme with bioluminescence, has emerged as a promising fluorophore due to its improved biocompatibility. However, the enzyme usually possesses weaker luminescence and stability relative to its chemically-based competitors. Here, we report a novel functional mutant luciferase with both enhanced luminescence and long-term serum stability. For the preparation of the modified Renilla luciferase, a new bacterial subcloning design was established. The luciferase coding DNA sequence was redesigned so that mutant luciferase could be easily expressed in an Escherichia coli system. The mutant Renilla luciferase, which we called "m-Rluc," demonstrated characteristic enzymatic functions and showed a 5.6-fold increase in luminescence activity. In addition, the enzyme's physiological stability remained >80% for more than 5days, in contrast to conventional luciferase, termed "hrluc," which disappeared within a few hours. We suggest that this novel biological luciferase probe may be a great tool for both in vitro and in vivo medical diagnostics.

The G-quadruplex augments translation in the 5' untranslated region of transforming growth factor ß2.

Transforming growth factor ß2 (TGFß2) is a versatile cytokine with a prominent role in cell migration, invasion, cellular development, and immunomodulation. TGFß2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition, angiogenesis, and immunosuppression. As it is well-documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at the post-transcriptional level. Bioinformatics analysis predicts an existence of a 23-nucleotide putative G-quadruplex sequence (PG4) in the 5' untranslated region (UTR) of TGFß2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using ultraviolet and circular dichroism spectroscopy. Footprinting studies further validated its existence in the presence of a neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using a dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in the context of the entire 5' UTR sequence interestingly enhances gene expression. Mutation or removal of the G-quadruplex sequence from the 5' UTR of the gene diminished the level of expression of this gene at the translational level. Thus, here we highlight an activating role of the G-quadruplex in modulating gene expression of TGFß2 at the translational level and its potential to be used as a target for the development of therapeutics against cancer.

Enhancement of gene silencing effect and membrane permeability by Peptide-conjugated 27-nucleotide small interfering RNA.

Two different sizes of siRNAs, of which one type was 21-nucleotide (nt) siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5'-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane permeability and intracellular accumulation.

Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives.

Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by 1H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%-50%, HPG4 showed 10-100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase.

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Kisspeptin directly excites anorexigenic proopiomelanocortin neurons but inhibits orexigenic neuropeptide Y cells by an indirect synaptic mechanism.

The neuropeptide kisspeptin is necessary for reproduction, fertility, and puberty. Here, we show strong kisspeptin innervation of hypothalamic anorexigenic proopiomelanocortin (POMC) cells, coupled with a robust direct excitatory response by POMC neurons (n > 200) to kisspeptin, mediated by mechanisms based on activation of a sodium/calcium exchanger and possibly opening of nonselective cation channels. The excitatory actions of kisspeptin on POMC cells were corroborated with quantitative PCR data showing kisspeptin receptor GPR54 expression in the arcuate nucleus, and the attenuation of excitation by the selective kisspeptin receptor antagonist, peptide 234. In contrast, kisspeptin inhibits orexigenic neuropeptide Y (NPY) neurons through an indirect mechanism based on enhancing GABA-mediated inhibitory synaptic tone. In striking contrast, gonadotropin-inhibiting hormone (GnIH and RFRP-3) and NPY, also found in axons abutting POMC cells, inhibit POMC cells and attenuate the kisspeptin excitation by a mechanism based on opening potassium channels. Together, these data suggest that the two central peptides that regulate reproduction, kisspeptin and GnIH, exert a strong direct action on POMC neurons. POMC cells may hypothetically serve as a conditional relay station downstream of kisspeptin and GnIH to signal the availability of energy resources relevant to reproduction.

Bunyamwera virus can repair both insertions and deletions during RNA replication.

The genomic termini of RNA viruses contain essential cis-acting signals for such diverse functions as packaging, genome translation, mRNA transcription, and RNA replication, and thus preservation of their sequence integrity is critical for virus viability. Sequence alteration can arise due to cellular mechanisms that add or remove nucleotides from terminal regions, or, alternatively, from introduction of sequence errors through nucleotide misincorporation by the error-prone viral RNA-dependent RNA polymerase (RdRp). To preserve template function, many RNA viruses utilize repair mechanisms to prevent accumulation of terminal alterations. Here we show that Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family of segmented negative-sense RNA viruses, also can repair its genomic termini. When an intact nontranslated region (NTR) was added to the anti-genomic 3' end, it was precisely removed, to restore both length and RNA synthesis function of the wild-type template. Furthermore, when nucleotides were removed from the anti-genome 3' end, and replaced with a duplicate and intact NTR, both the external NTR were removed, and the missing nucleotides were restored, thus, indicating that the BUNV RdRp can both remove and add nucleotides to the template. We show that the mechanism for repair of terminal extensions is likely that of internal entry of the viral RdRp during genome synthesis. Possible mechanisms for repair of terminal deletions are discussed.

Well-defined poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) vectors with low toxicity and high gene transfection efficiency.

Successful gene delivery vectors for clinical translation should have high transfection efficiency and minimal toxicity. In this work, well-defined poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) (PGEA) vectors with flanking cationic secondary amine and nonionic hydroxyl units were prepared via the ring-opening reaction of the pendant epoxide groups of poly(glycidyl methacrylate) with the amine moieties of ethanolamine. It was found that PGEA carriers possess very low toxicity (<10% of the toxicity of branched polyethylenimine (PEI, 25 kDa), while exhibiting surprisingly excellent transfection efficiency (higher than or comparable to that of PEI (25 kDa)) in different cell lines. A series of transfection and cytotoxicity assays revealed that PGEAs are highly promising as a new class of safe and efficient gene delivery vectors for future clinical gene therapies.

Antibody profiling by Luciferase Immunoprecipitation Systems (LIPS).

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log(10) for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons.

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.