Presence Of Coelenterazine Research Articles

Abstract 706: Rapid detection of SARS-CoV-2 antigen and antibody seroconversion in clinical specimens using a novel Surface Programmable Activation Receptor (SPAR) modified T cell diagnostic method

Abstract Infection by SARS-CoV-2 virus or past exposure to COVID19, is of central concern to the management of cancer patient co-morbidities. Currently available testing methods require separate technologies to determine if a patient has anti- SARS-CoV-2 antibodies or if they are currently infected. A rapid, reliable, dual-use diagnostic platform deployed near the cancer patient would make a critical contribution to their care. We previously developed a biomarker detection system using modified Jurkat-T cells engineered into specific and sensitive biosensors that display a wide dynamic range with low background signal. In this system, the T cell receptor (TCR) is fused to an antibody binding domain (mouse FcγRI) and the cells are also engineered to express a high level of the calcium dependent photoprotein, aequorin. The modified TCR can be programmed for detection of a target of interest by addition of specific and appropriately tagged target detection molecules (TDMs) which bind the FcγRI.Using this system, we have developed novel assays specific for detection of SARS-CoV-2 antigen or serum antibodies against SARS-CoV-2 in clinical specimens, using mouse IgG2a-tagged TDMs provided by Twist Bioscience. For antigen detection, mouse IgG2a-Fc tagged SARS-CoV-2 spike antibody (IgG) was used as the assay TDM, sensitizing the cells to SARS-CoV-2 spike protein. For antibody detection, mouse IgG2a-Fc tagged SARS-CoV-2 spike protein (S1 domain) was used as the assay TDM with T cell activation caused by binding to serum antibodies against SARS-CoV-2 spike protein. Following binding of either target, T cell activation causes a rapid release of calcium in the modified T cells, leading to a rapid light emission by aequorin in the presence of coelenterazine, CTZ. This light emission is detectable in less than 5 minutes using a simple luminometer, and with no specimen pre-treatment. Results from both contrived and clinical specimens using each diagnostic method will be presented to demonstrate their accuracy, reproducibility, sensitivity, specificity, and speed. Results are highly reproducible at sub-nanomolar spike protein concentrations in viral transport media (VTM) and human serum, respectively. Real-world clinical seropositive specimens from convalescent COVID-19 patients as well as serum from patients exposed to related viruses or other disease states were tested using the antibody detection assay. Gamma-irradiated clinical SARS-CoV-2 isolate as well as related and unrelated viral isolates on nasal swabs were tested using the antigen detection assay. Citation Format: Aaron K. LeFebvre, Melissa Frenchmeyer, Kyle M. McQuaid, MRussell Williams, Charles M. McBrairty, Joseph D. Kittle. Rapid detection of SARS-CoV-2 antigen and antibody seroconversion in clinical specimens using a novel Surface Programmable Activation Receptor (SPAR) modified T cell diagnostic method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 706.

Listeria innocua Dps as a nanoplatform for bioluminescence based photodynamic therapy utilizing Gaussia princeps luciferase and zinc protoporphyrin IX

Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490 nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent.

Differential Requirements of Hsp90 and DNA for the Formation of Estrogen Receptor Homodimers and Heterodimers

The two estrogen receptor (ER) subforms, ERalpha and ERbeta, are capable of forming DNA-binding homodimers and heterodimers. Although binding to DNA is thought to stabilize ER dimers, how ERalpha/alpha, ERbeta/beta, and ERalpha/beta dimerization is regulated by DNA and the chaperone protein Hsp90 is poorly understood. Using our highly optimized bioluminescence resonance energy transfer assays in conjunction with assays for transcriptional activation of ERs, we determined that DNA binding appears to play a minor role in the stabilization of ER dimers, especially in the case of ERbeta/beta homodimers. These findings suggest that ER dimers form before they associate with chromatin and that DNA binding plays a minor role in stabilizing ER dimers. Additionally, although Hsp90 is essential for the proper dimerization of ERalpha/alpha and ERalpha/beta, it is not required for the proper dimerization of ERbeta/beta. Despite this, Hsp90 is critical for the estrogen-dependent transcriptional activity of the ERbeta/beta homodimer. Thus, Hsp90 is implicated as an important regulator of distinct aspects of ERalpha and ERbeta action.

Screening for Unknown Mutations by a Bioluminescent Protein Truncation Test with Homogeneous Detection

The protein truncation test (PTT) is important in screening for unknown mutations that cause premature termination of mRNA translation. PTT involves amplification of the interrogated sequence, in vitro transcription/translation, separation of the generated polypeptides, and detection. In this article, we report a bioluminescent protein truncation test, in which the detection of the nascent protein is performed directly in the expression mixture, within seconds, without the need for separation and purification. A DNA fragment encoding apoaequorin is fused, in-frame, downstream of the interrogated sequence. The fusion product is subjected to in vitro, coupled transcription and translation in the presence of coelenterazine. A wild-type DNA template allows translation to continue after the 3' end of the interrogated sequence, producing a chimeric protein whose C-terminal domain is the photoprotein aequorin. Aequorin is detected, with a high sensitivity, by its characteristic Ca(2+)-triggered, flash-type bioluminescent reaction. Active photoprotein is not produced when a truncating mutation is present in the interrogated sequence. As a model, the method was applied to the detection of truncating mutations in the APC gene (adenomatous polyposis coli).

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

Signal transduction leading to low-temperature tolerance in Arabidopsis thaliana.

Calcium is used by most cells to convert external signals into biochemical events within the cytosol. To detect the effects of cold stress, a gene encoding apoaequorin has been introduced into various cell types that, in the presence of coelenterazine, allows calcium levels to be monitored by the emission of blue light. All cell types respond to the cold by elevating calcium. This event is rapid after cold stress but then shows a slower kinetic response. The magnitude is dependent on both the rate and the final temperature to which cooling occurs. It would appear that calcium is transferred both from outside the cell and from the vacuole into the cytosol. The more rapid phase involves the former and the slower phase the latter. By studying mutant plants, it has been identified that an increase in intracellular calcium can activate the expression of transcription factors that control the subsequent transcription of a whole battery of genes which must be switched on to provide cold tolerance in the plant.

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Cotranslational Formation of Active Photoprotein Obelin in a Cell-Free Translation System: Direct Ultrahigh Sensitive Measure of the Translation Course

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity ofin vitrotranslation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.

Presence of coelenterazine in mesopelagic fishes from the Strait of Messina

Coelenterazine, a luciferine, and luciferase activity specific to coelenterazine were detected and assayed in various tissues of mid-water fishes, Argyropelecus hemigymnus, Chauliodus sloani, Myctophum punctatum, Vinciguerria attenuata and Cyclothone braueri. Coelenterazine was found mainly in digestive systems and photophores of these fishes, A large species to species variation was found: extremely high levels of coelenterazine and luciferase were found in the M. punctatum digestive system whereas only a very low level of coelenterazine was detected in C., braueri. Coelenterazine was detected in A. hemigymnus eggs supporting the hypothesis of a maternal transfer of luminous capabilities. Luciferase activity specific to coelenterazine was found in photophores as well as in various other tissues suggesting another (besides light emission) biological function for this enzymatic activity, Distribution of coelenterazine in all tissues of the individuals supports the hypothesis of the dietary acquisition of coelenterazine by these fishes.

Presence of coelenterazine in non-bioluminescent marine organisms

1. 1. Coelenterazine is known to be widely distributed in bioluminescent marine organisms, as a luciferin and as the functional group of photoproteins. 2. 2. The present data indicate that coelenterazine is also present in various non-bioluminescent marine organisms. 3. 3. In most of the non-bioluminescent organisms, coelenterazine is probably absorbed from ingested food.