A genetically encoded photosensitizer

Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to beta-galactosidase and phospholipase Cdelta1 pleckstrin homology domain.     

Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.     

Structural basis for the phototoxicity of the fluorescent protein KillerRed

The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by “pre-bleached” KillerRed.     

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the β-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu⁶⁸ and Ser¹¹⁹, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.     

Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.     

Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed

The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.     

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.     

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.     

Light-stimulated phosphorylation of proteins in cell-free extracts from Trichoderma viride

When illuminated by visible light, cell-free extracts from the fungus Trichoderma viride catalysed the phosphorylation of at least two proteins with molecular masses of 18 and 114 kDa which were practically absent when the phosphorylation was performed in the dark. The effect of light could be substituted by 3mM cyclic AMP, not only in the cell-free extract, but also in the separated cytosol. It is concluded that the process of photoinduced conidiation in Trichoderma involves phosphorylation of conidiation-specific proteins by (a) cyclic AMP-dependent protein kinase(s) present in the cytosol.     

Positive selection vector using the KillerRed gene

Plasmid pZK18S is a novel positive selection vector containing the genetically encoded photosensitizer KillerRed as the selection marker. When transformed into host cells (lacI^q or lacI⁺), the common medical surgical light is sufficient to activate phototoxicity of KillerRed. Thus, only the recombinants with disrupted reading frame of KillerRed genes finally allow forming viable colonies. Because lethality of KillerRed relies on light irradiation, no special host and culture medium are required to amplify and prepare pZK18S vector in larger quantities. The pZK18S was reliable and highly efficient for constructing the serial analysis of gene expression (SAGE) library.     

Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin

The 48-kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5-10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48-kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP-binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP-binding protein thereby displaces 48-kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.     

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

Background

Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.

Methods

HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo.

Results

Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG.

Conclusions

miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor.

General significance

This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.     

Fluorescent proteins: maturation, photochemistry and photophysics

It has long been appreciated that green fluorescent protein (GFP) autocatalytically forms its chromophore in a host-independent process; several of the initial steps in the reaction have recently been elucidated. Nevertheless, the end points of the process are unexpectedly diverse, as six chemically distinct chromophores, including two with three rings, have been identified. All fluorescent proteins continuously produce a low level of reactive oxygen species under illumination, which, in some cases, can lead to host cell death. In one extreme but useful example, the red fluorescent protein KillerRed can be used to selectively destroy cells upon brief illumination. Finally, when photophysical processes such as excited-state proton transfer, reversible photobleaching and photoactivation are understood, useful research tools, for example, real-time biosensors and optical highlighters, can result; however, side effects of their use may lead to significant artifacts in time-dependent microscopy experiments.     

Biosynthesis of the light-harvesting chlorophyll a/b protein. Control of messenger RNA activity by light

1. Antibodies raised against the 26000-Mr polypeptides of the light-harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000-Mr polypeptides synthesized when pea leaf poly(A)-containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000-Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light-harvesting chlorophyll a/b complex. 2. The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light-harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark-grown plants. Exposure to continuous illumination caused these levels to increase by at least ten-fold and led to the appearance of large quantities of the light-harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light-harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants). 3. Messenger RNAs encoding the light-harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat-germ 100 000 X g supernatant "run-off" system, to form light-harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light-harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs. 4. Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light-harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000-Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000-Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light-harvesting chlorophyll a/b protein fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light-harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.     

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light‐dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm² and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm² and light doses of 140 J/cm², 170 J/cm² and 200 J/cm². Irradiation of KillerRed‐expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP‐like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed.

Representative fluorescence image of two KillerRed‐expressing spheroids before and immediately after CW irradiation.     

T vector bearing KillerRed protein marker for red/white cloning screening

As a novel selection marker for DNA cloning, the genetically encoded photosensitizer KillerRed was used to achieve red/white cloning screening within the pZK18T T-vector system. KillerRed functioned without cofactors, inducers, or substrates. KillerRed-based red/white cloning screening was reliable in that bacteria containing DNA inserts that disrupt functional KillerRed expression form white colonies or red colonies as backgrounds. KillerRed simplifies assembly of customized vector and recombinant screening procedures. No special host or culture medium is required to amplify and prepare the vector in larger quantities. It makes high-throughput general-purpose cloning screening simpler, less expensive, and more effective.     

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.     

Rhodamine immunohistofluorescence applied to plant tissue.

Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia, Scop.) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence.