- Proceedings of the National Academy of Sciences of the United States of America
- Published over 3 years ago
Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.
Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.
Bioluminescent imaging (BLI) has been widely applicable in the imaging of process envisioned in life sciences. As the most conventional technique for BLI, the firefly luciferin-luciferase system is exceptionally functional in vitro and in vivo. The state-of-the-art strategy in such a system is to cage the luciferin, in which free luciferin is conjugated with distinctive functional groups, thus accommodating an impressive toolkit for exploring various biological processes, such as monitoring enzymes activity, detecting bioactive small molecules, evaluating the properties of molecular transporters, etc. This review article summarizes the rational design of caged luciferins towards diverse biotargets, as well as their applications in bioluminescent imaging. It should be emphasized that these caged luciferins can stretch out the applications of bioluminescence imaging and shed light upon understanding the pathogenesis of various diseases.
- Journal of photochemistry and photobiology. B, Biology
- Published almost 5 years ago
Firefly luciferase is the most important and studied bioluminescence system. Due to very interesting characteristics, this system has gained numerous biomedical, pharmaceutical and bioanalytical applications, among others. In order to improve the use of this system, various researchers have tried to understand experimentally the colour of bioluminescence, and to create ways of tuning the colour emitted. The objective of this manuscript is to review the experimental studies of firefly luciferin and oxyluciferin, and related analogues, fluorescence and bioluminescence.
Split reporter proteins capable of self-association and reactivation have applications in biomedical research, but designing these proteins, especially the selection of appropriate split points, has been somewhat arbitrary. We describe a new methodology to facilitate generating split proteins using split GFP as a self-association module. We first inserted the entire GFP module at one of several candidate split points in the protein of interest, and chose clones that retained the GFP signal and high activity relative to the original protein. Once such chimeric clones were identified, a final pair of split proteins was generated by splitting the GFP-inserted chimera within the GFP domain. Applying this strategy to Renilla reniformis luciferase, we identified a new split point that gave 10 times more activity than the previous split point. The process of membrane fusion was monitored with high sensitivity using a new pair of split reporter proteins. We also successfully identified new split points for HaloTag protein and firefly luciferase, generating pairs of self-associating split proteins that recovered the functions of both GFP and the original protein. This simple method of screening will facilitate the designing of split proteins that are capable of self-association through the split GFP domains.
Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.
Bioluminescent microcapsules uploading D-luciferin have been fabricated by using the covalent assembly of firefly luciferase and alginate dialdehyde through a layer-by-layer technique. Such assembled microcapsules can produce visible light in the region of 520-680 nm, which can activate the photosensitizers rose bengal (RB) and hypocrellin B (HB) after adding ATP. The microcapsules uploading photosensitizers (RB or HB) have an obvious property to prevent the proliferation of tumor cells in the dark. The assembled bioluminescent microcapsules can be potentially used as photon donors for bioimaging, ATP detection, and photodynamic therapy.
Firefly luciferase adenylates and oxidizes d-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. d-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of d-luciferin in live luciferase-expressing cells. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.
The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive imaging in live mice. However, photon emission from the luciferase is crucially dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of bioluminescence imaging.