ABSTRACT
The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.
Subject(s)
Fireflies , Peroxisomal Targeting Signals , Male , Animals , Fireflies/genetics , Fireflies/metabolism , Peroxisomal Targeting Signals/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Luciferases/genetics , Luciferases/metabolism , Base SequenceABSTRACT
Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.
Subject(s)
Fireflies , Islets of Langerhans Transplantation , Animals , Rats , Fireflies/metabolism , Luciferases , Animals, Genetically Modified , Diagnostic Imaging , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Luminescent MeasurementsABSTRACT
BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.
Subject(s)
Fireflies , Glioma , Animals , Child , Humans , Young Adult , Fireflies/metabolism , Proto-Oncogene Proteins B-raf , Glioma/drug therapy , Glioma/genetics , Glioma/metabolism , Treatment Outcome , Mutation , Mitogen-Activated Protein Kinases , Oximes , Pyridones , Pyrimidinones/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic useABSTRACT
"Firefly rats" ubiquitously express the luciferase reporter gene under the control of constitutively active ROSA26 promoter in inbred Lewis rats. Due to the minimal immunogenicity of luciferase, wide applications of Firefly rats have been reported in solid organ/cell transplantation studies for in vivo imaging, permitting quantitative and non-invasive tracking of the transplanted graft. ROSA26 is a non-coding gene and generally does not affect the expression of other endogenous genes. However, the effect of ubiquitous luciferase expression on islet morphology and function has not been thoroughly investigated, which is critical for the use of Firefly rats as islet donors in islet transplantation studies. Accordingly, in vivo glucose homeostasis (i.e., islet function in the native pancreas) was compared between age-matched luciferase-expressing Firefly rats and non-luciferase-expressing rats. In vivo assessments demonstrated no statistical difference between these rats in non-fasting blood glucose levels, intraperitoneal glucose tolerance tests, and glucose-stimulated serum C-peptide levels. Furthermore, islets were isolated from both rats to compare the morphology, function, and metabolism in vitro. Isolated islets from both rats exhibited similar in vitro characteristics in post-isolation islet yield, islet size, beta cell populations, insulin content per islet, oxygen consumption rate, and glucose-stimulated insulin secretion. In conclusion, ubiquitous luciferase expression in Firefly rats does not affect their islet morphology, metabolism, and function; this finding is critical and enables the use of isolated islets from Firefly rats for the dual assessment of islet graft function and bioluminescence imaging of islet grafts.
Subject(s)
Islets of Langerhans Transplantation , Islets of Langerhans , Rats , Animals , Fireflies/metabolism , Rats, Inbred Lew , Islets of Langerhans/metabolism , Insulin/metabolism , Glucose/pharmacology , Glucose/metabolism , Luciferases , Blood Glucose/metabolismABSTRACT
The quantum yield (Q) of the "cold light" of firefly bioluminescence (BL) is remarkably high due to its nonradiative decay is extremely minimized. Thus, an artificial firefly represents the new generation of biomimetic "cold light" source with highest energy utilization. However, to manufacture a firefly-biomimetic "cold light" in vitro, one has to overcome several challenges including realization of the firefly BL cycle by incorporating the two important enzymes (i.e., firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE)) in one system. Here in this work, using self-prepared Fluc, LRE, and the main substrates, we realized the firefly BL cycle both in vitro and in cells. Moreover, using combinational analyses of HPLC and nESI-CID-MS/MS, we identified the main chemicals in the metabolic pathways underlying the firefly BL cycle. Using theoretical simulations, we revealed an optimum chemical route which balances the reaction cycle to achieve the highest BL intensity with the least chemical supplies. We anticipate that this pioneering study of the firefly cycle would provide industry with the opportunity to design tunable, economical, biomimetic "cold light" device in near future.
Subject(s)
Biosensing Techniques , Fireflies , Animals , Fireflies/metabolism , Tandem Mass Spectrometry , Luciferases, Firefly , Luminescent MeasurementsABSTRACT
This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.
Subject(s)
Decapodiformes , Fireflies , Albumins/metabolism , Albuminuria/diagnosis , Animals , Decapodiformes/chemistry , Fireflies/metabolism , Firefly Luciferin/metabolism , Humans , LuciferinsABSTRACT
Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.
Subject(s)
Fireflies , Luminescence , Animals , Fireflies/genetics , Fireflies/metabolism , Genes, Reporter , Indicators and Reagents , Luciferases/genetics , Luciferases/metabolism , Luciferases, Firefly/geneticsABSTRACT
Among all bioluminescent organisms, the firefly is the most famous, with a high luminescent efficiency of 41%, which is widely used in the fields of biotechnology, biomedicine and so on. The entire bioluminescence (BL) process involves a series of complicated in-vivo chemical reactions. The BL is initiated by the enzymatic oxidation of luciferin (LH2). However, the mechanism of the efficient spin-forbidden oxygenation is far from being totally understood. Via MD simulation and QM/MM calculations, this article describes the complete process of oxygenation in real protein. The oxygenation of luciferin is initiated by a single electron transfer from the trivalent anionic LH2 (L3-) to O2 to form 1[Lâ¢2- O2â¢-]; the entire reaction is carried out along the ground-state potential energy surface to produce the dioxetanone (FDO-) via three transition states and two intermediates. The low energy barriers of the oxygenation reaction and biradical annihilation involved in the reaction explain this spin-forbidden reaction with high efficiency. This study is helpful for understanding the BL initiation of fireflies and the other oxygen-dependent bioluminescent organisms.
Subject(s)
Fireflies/metabolism , Luciferases, Firefly/metabolism , Luminescent Agents/metabolism , Animals , Heterocyclic Compounds, 1-Ring/metabolism , Luminescence , Luminescent Measurements/methods , Oxidation-ReductionABSTRACT
Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (D-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert L-luciferin to D-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.
Subject(s)
Biological Evolution , Fireflies/genetics , Firefly Luciferin/metabolism , Animals , Fireflies/metabolism , Proteome , Species Specificity , TranscriptomeABSTRACT
The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.
Subject(s)
Genes, Reporter/genetics , Genetic Vectors/genetics , Polymerase Chain Reaction/methods , Animals , Cell Line, Tumor , Efficiency/physiology , Fireflies/metabolism , Green Fluorescent Proteins/metabolism , Luciferases/metabolism , Plasmids/genetics , Promoter Regions, Genetic/genetics , Transfection/methods , Transgenes/genetics , Zebrafish/metabolismABSTRACT
Metabolite identification is the greatest challenge when analysing metabolomics data, as only a small proportion of metabolite reference standards exist. Clustering MS/MS spectra is a common method to identify similar compounds, however interrogation of underlying signature fragmentation patterns within clusters can be problematic. Previously published high-resolution LC-MS/MS data from the bioluminescent beetle (Photinus pyralis) provided an opportunity to mine new specialized metabolites in the lucibufagin class, compounds important for defense against predation. We aimed to 1) provide a workflow for hierarchically clustering MS/MS spectra for metabolomics data enabling users to cluster, visualise and easily interrogate the identification of underlying cluster ion profiles, and 2) use the workflow to identify key fragmentation patterns for lucibufagins in the hemolymph of P. pyralis. Features were aligned to their respective MS/MS spectra, then product ions were dynamically binned and resulting spectra were hierarchically clustered and grouped based on a cutoff distance threshold. Using the simplified visualization and the interrogation of cluster ion tables the number of lucibufagins was expanded from 17 to a total of 29.
Subject(s)
Fireflies/metabolism , Hemolymph/metabolism , Steroids/metabolism , Animals , Chromatography, Liquid/methods , Cluster Analysis , Metabolomics/methods , Tandem Mass SpectrometryABSTRACT
We analyzed the near-degenerate states of the firefly dioxetanone anion (FDO-) and its prototypes, especially in the biradical region, using multi-configurational approaches. The importance of utilizing full valence active spaces by means of density-matrix renormalization group self-consistent field (DMRG-SCF) calculations was described. Our results revealed that the neglect of some valence orbitals can affect the quantitative accuracy in later multi-reference calculations or the qualitative conclusion when optimizing conical intersections. Using all of the relevant valence orbitals of FDO-, we confirmed that there were two conical intersections, as reported in previous work, and that the intersecting states were changed when the active space was enlarged. Beyond these, we found that there were strong interactions between states in the biradical regions, in which the changes in entanglements can be used to visualize the interacting state evolution.
Subject(s)
Fireflies/chemistry , Heterocyclic Compounds, 1-Ring/chemistry , Animals , Anions/chemistry , Fireflies/metabolism , Luminescence , Quantum Theory , Thiazoles/chemistryABSTRACT
The Genji firefly, Luciola cruciata, is widely distributed throughout the major Japanese islands (Honshu, Shikoku, and Kyushu) and distinguished into two ecological types on the basis of the flash interval of the mate-seeking males (4-sec slow-flash or 2-sec fast-flash intervals). The boundary of the ecological types corresponds to the Fossa Magna, a great rupture zone that separates eastern and western Japan. Although the degree of genetic differentiation of the two types has been evaluated using allozyme and mitochondrial DNA sequence data, it has not been evaluated using genome-wide data. Based on the genome-wide data obtained using single-end restriction-site-associated DNA (RAD-Seq), principal component, gene-level phylogenetic tree, admixture, and Wright's fixation index analyses, we identified three phylogenetic groups in L. cruciata: East-Honshu, West-Honshu, and Kyushu. This grouping corresponds to the ecological types: East-Honshu to the slow-flash type and West-Honshu and Kyushu to the fast-flash type. Although introgression was exceptionally observed around adjacent or artificially transplanted areas, gene flow among the groups was almost absent in the natural populations. The phylogenetic tree under the coalescent model also evaluated differentiation among the East-Honshu, West-Honshu and Kyushu groups. Furthermore, because the distribution patterns of the three groups are consistent with the geological history of Japanese islands, a vicariant speciation scenario of L. cruciata is concluded. In addition, we identified genetic markers that can be used to distinguish the three genetic groups for genetic management of firefly transplantation in nature conservation and regeneration.
Subject(s)
Fireflies/genetics , Fireflies/metabolism , Animals , Base Sequence/genetics , DNA, Mitochondrial/genetics , Demography , Genetic Variation/genetics , Genome-Wide Association Study/methods , Japan , Phylogeography/methods , Population DensityABSTRACT
Tepuis are Precambrian sandstone tabletop mountains in South America that can reach up to â¼3,000 m in elevation. Their highest summits are both physiographically and ecologically isolated from the surrounding upland savannah and lush tropical rainforest, and they face particularly hostile, challenging environmental conditions [1,2] (Figure 1A). Taxa thriving on high tepui summits must adapt to resource-limited and highly competitive ecosystems. The toad genus Oreophrynella is exclusively found on tepui slopes and summits [3] and is particularly well-adapted to the tepui top environment. We here report on an unanticipated and possibly large-scale predation on this endemic toad by non-bioluminescent firefly larvae.
Subject(s)
Bufonidae/parasitology , Fireflies/parasitology , Altitude , Animals , Anura , Bufonidae/physiology , Ecology , Ecosystem , Fireflies/metabolism , Larva/parasitology , Phylogeny , Predatory Behavior/physiology , Rainforest , South AmericaABSTRACT
The Bermuda fireworm Odontosyllis enopla exhibits an extremely tight circalunar circadian behavior that results in an impressive bioluminescent mating swarm, thought to be due to a conventional luciferase-mediated oxidation of a light-emitting luciferin. In addition, the four eyes become hypertrophied and heavily pigmented, and the nephridial system is modified to store and release gametes and associated secretions. In an effort to elucidate transcripts related to bioluminescence, circadian or circalunar periodicity, as well as epitoky-related changes of the eyes and nephridial system, we examined the transcriptomic profile of three female O. enopla during a bioluminescent swarm in Ferry Reach, Bermuda. Using the well-characterized luciferase gene of the Japanese syllid Odontosyllis undecimdonta as a reference, a complete best-matching luciferase open reading frame (329 amino acids in length) was found in all three individuals analyzed in addition to numerous other paralogous sequences in this new gene family. No photoproteins were detected. We also recovered a predicted homolog of 4-coumarate-CoA ligase (268 amino acids in length) that best matched luciferase of the firefly Luciola with the best predicted template being the crystal structure of luciferase for Photinus pyralis, the common eastern firefly. A wide variety of genes associated with periodicity were recovered including predicted homologs of clock, bmal1, period, and timeless. Several genes corresponding to putative epitoky-related changes of the eyes were recovered including predicted homologs of a phototransduction gene, a retinol dehydrogenase and carotenoid isomerooxygenase as well as a visual perception related retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase. Genes correlating to putative epitoky-related changes of the nephridia included predicted homologs of nephrocystin-3 and an egg-release sex peptide receptor.
Subject(s)
Annelida/genetics , Luciferases/genetics , Amino Acid Sequence , Animals , Circadian Rhythm/genetics , Fireflies/metabolism , Luciferases, Firefly/genetics , Luminescent Measurements , Phylogeny , Polychaeta/metabolism , Sexual Maturation , TranscriptomeABSTRACT
Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.
Subject(s)
Fireflies/metabolism , Firefly Luciferin/metabolism , Hydroquinones/metabolism , Animals , Chromatography, High Pressure Liquid , Deuterium/chemistry , Fireflies/growth & development , Firefly Luciferin/analysis , Hydroquinones/chemistry , Isotope Labeling , Japan , Pupa/metabolism , Stereoisomerism , Tandem Mass SpectrometryABSTRACT
Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Adenosine Triphosphate/metabolism , Animals , Escherichia coli/metabolism , Fireflies/metabolism , Humans , Protein Folding , Saccharomyces cerevisiae/metabolismABSTRACT
Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.
Subject(s)
Fireflies/metabolism , Firefly Luciferin/metabolism , Luciferases/metabolism , Palmitoyl-CoA Hydrolase/metabolism , Animals , Luminescence , StereoisomerismABSTRACT
The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns-ps) fluorescence spectroscopy, NMR, X-ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high-energy species that undergoes decarboxylation to form directly the product in its S1 state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ "antenna proteins," lumazine protein and the green-fluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca2+ -regulated photoproteins and the antenna protein interactions.
Subject(s)
Luminescent Proteins/metabolism , Animals , Bacteria/metabolism , Carboxylic Acids/metabolism , Crystallography, X-Ray , Fireflies/metabolism , Luminescent Measurements , Luminescent Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Spectrometry, FluorescenceABSTRACT
Postcopulatory sexual selection is recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, the molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. During mating, male Photinus fireflies transfer to females a spermatophore gift manufactured by multiple reproductive glands. Here we combined transcriptomics of both male and female reproductive glands with proteomics and metabolomics to better understand the synthesis, composition and fate of the spermatophore in the common Eastern firefly, Photinus pyralis. Our transcriptome of male glands revealed up-regulation of proteases that may enhance male fertilization success and activate female immune response. Using bottom-up proteomics we identified 208 functionally annotated proteins that males transfer to the female in their spermatophore. Targeted metabolomic analysis also provided the first evidence that Photinus nuptial gifts contain lucibufagin, a firefly defensive toxin. The reproductive tracts of female fireflies showed increased gene expression for several proteases that may be involved in egg production. This study offers new insights into the molecular composition of male spermatophores, and extends our understanding of how nuptial gifts may mediate postcopulatory interactions between the sexes.