Genetically targeted chromophore-assisted light inactivation

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.     

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.     

Potent DNA photocleavage by zinc(II) complexes of cationic bis-porphyrins linked with aliphatic diamine

We have prepared zinc(II) complexes of cationic bis-porphyrins, as one of the attempts to improve less DNA photocleavage activities of the metal-free bis-porphyrins composed of two H(2)TMPyP-like chromophores, linked with a series of aliphatic diamines. The less activities seemed to be derived from their intermolecular self-aggregation properties in aqueous solution. The zinc(II) insertion into the metal-free cationic bis-porphyrins completely removed their self-aggregation properties, most probably due to steric hindrance between axial ligands of zinc(II) chromophores of the cationic bis-porphyrins. The DNA photocleavage activities of the zinc(II) complexes were fully enhanced, which were three times larger than that of the lead compound H(2)TMPyP. Quantitative analysis of singlet oxygen production by photosensitization of cationic bis-porphyrins was performed using 1,3-diphenylisobenzofuran, and the singlet oxygen productivities of them were found to be related to their solution properties. There is a good relationship between the activities and the productivities, which will provide insights into the further development of more effective DNA photocleavage agents.     

Chromophore-assisted laser inactivation (CALI) to elucidate cellular mechanisms of cancer.

Chromophore-assisted laser inactivation (CALI) is a new technology for acute protein inactivation in living cells. It targets laser energy to specific proteins via non-function-blocking antibodies that are labeled with the dye malachite green. Excitation of the dye generates short-lived free radicals that damage the bound protein without affecting other cellular components. The wavelength of laser light used (620 nm) is not readily absorbed by cells such that non-specific light damage does not occur. CALI provides an alternative to other inactivation strategies and has the advantages of high spatial and temporal resolution. The ultimate value of this technology for cancer research will be assessed by how effective CALI is in ascribing in situ function during cancer-relevant processes and in identifying and validating protein targets for drug discovery. Recent work using CALI on ezrin and pp60-c-src, two proteins that may be involved in cancer, suggests its potential. Further application of CALI will likely be of utility for understanding cellular mechanisms of cancer and developing cancer therapeutics.     

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.     

Adapting chromophore-assisted laser inactivation for high throughput functional proteomics.

Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.     

Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as β-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease.     

Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin

For the purpose of the basic study of photodynamic therapy, the activity of the water-soluble P(V)porphyrin, dimethoxyP(V)tetraphenylporphyrin chloride (DMP(V)TPP), on photosensitized protein damage was examined. The quantum yield of singlet oxygen generation by DMP(V)TPP (0.64) was comparable with that of typical porphyrin photosensitizers. Absorption spectrum measurement demonstrated the binding interaction between DMP(V)TPP and human serum albumin, a water-soluble protein. Photo-irradiated DMP(V)TPP damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of DMP(V)TPP by human serum albumin supported the electron transfer mechanism. The estimated contribution of the electron transfer mechanism is 0.64. These results suggest that the activity of DMP(V)TPP can be preserved under lower oxygen concentration condition such as tumor.     

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

Porphyrin depth in lipid bilayers as determined by iodide and parallax fluorescence quenching methods and its effect on photosensitizing efficiency

Photosensitization by porphyrins and other tetrapyrrole chromophores is used in biology and medicine to kill cells. This light-triggered generation of singlet oxygen is used to eradicate cancer cells in a process dubbed "photodynamic therapy," or PDT. Most photosensitizers are of amphiphilic character and they partition into cellular lipid membranes. The photodamage that they inflict to the host cell is mainly localized in membrane proteins. This photosensitized damage must occur in competition with the rapid diffusion of singlet oxygen through the lipid phase and its escape into the aqueous phase. In this article we show that the extent of damage can be modulated by employing modified hemato- and protoporphyrins, which have alkyl spacers of varying lengths between the tetrapyrrole ring and the carboxylate groups that are anchored at the lipid/water interface. The chromophore part of the molecule, and the point of generation of singlet oxygen, is thus located at a deeper position in the bilayer. The photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. The vertical insertion of the sensitizers was assessed by two fluorescence-quenching techniques: by iodide ions that come from the aqueous phase; and by spin-probe-labeled phospholipids, that are incorporated into the bilayer, using the parallax method. These methods also show that temperature has a small effect on the depth when the membrane is in the liquid phase. However, when the bilayer undergoes a phase transition to the solid gel phase, the porphyrins are extruded toward the water interface as the temperature is lowered. These results, together with a previous publication in this journal, represent a unique and precedental case where the vertical location of a small molecule in a membrane has an effect on its membranal activity.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Singlet oxygen-mediated damage to proteins and its consequences

Proteins comprise approximately 68% of the dry weight of cells and tissues and are therefore potentially major targets for oxidative damage. Two major types of processes can occur during the exposure of proteins to UV or visible light. The first of these involves direct photo-oxidation arising from the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. Singlet oxygen can also be generated by a range of other enzymatic and non-enzymatic reactions including processes mediated by heme proteins, lipoxygenases, and activated leukocytes, as well as radical termination reactions. This paper reviews the data available on singlet oxygen-mediated protein oxidation and concentrates primarily on the mechanisms by which this excited state species brings about changes to both the side-chains and backbone of amino acids, peptides, and proteins. Recent work on the identification of reactive peroxide intermediates formed on Tyr, His, and Trp residues is discussed. These peroxides may be important propagating species in protein oxidation as they can initiate further oxidation via both radical and non-radical reactions. Such processes can result in the transmittal of damage to other biological targets, and may play a significant role in bystander damage, or dark reactions, in systems where proteins are subjected to oxidation.     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Biological inactivation by singlet oxygen: distinguishing O2(1 delta g) and O2(1 sigma g+)

Experiments were performed to determine whether bacterial inactivation in the separated-surface-sensitizer system for singlet oxygen generation is due to O2(1 delta g) or O2(1 sigma g+). The rates of inactivation of Gram-negative Salmonella typhimurium LT-2 and a nonpigmented strain of Gram-positive Sarcina lutea were found to increase linearly with the concentration of 1 delta g. The gas phase lifetime of the inactivating agent was found to be within the range of values expected for the gas phase lifetime of 1 delta g rather than 1 sigma g+. These measurements conclusively demonstrate that bacterial inactivation in this system is due predominantly to 1 delta g. Therefore, studies of bacterial inactivation with this singlet oxygen generating system can be used to assess the role of singlet oxygen in various biological and medically relevant situations.     

Functional proteomics using chromophore-assisted laser inactivation

Proteins are the molecules that fulfil most cellular functions and represent over 90% of drug targets in the market. Chromophore-assisted laser inactivation (CALI) provides a timely and locally restricted protein inactivation and has proven to specifically destroy protein function using dye-coupled ligands and laser irradiation. CALI involves the generation of short-lived radicals thus limiting the radius of covalent modifications to spatially restricted sites on the target molecule. A transient functional inactivation occurs if the radicals modify amino acids of the target protein that are responsible for function. Here we show specific inactivation of several protein targets, that are members of relevant signal transduction pathways. For each of these targets, simple and high throughput screening-scaleable assays have been developed, making it possible to quantify the observed inactivation. Activities of target proteins have been addressed in cell-free as well as cell-based assays employing human primary and tumor-derived cell lines. In all cases, at least 50% inactivation was achieved. The data presented here demonstrate that CALI is a highly versatile tool for validating disease relevant targets at the protein level. This approach also takes into account post-translational modifications like phosphorylation, glycosylation or acylation, thereby enlarging its applicability for many different types of targets.     

Interactions of dietary carotenoids with singlet oxygen (1O2) and free radicals: potential effects for human health

The dietary carotenoids provide photoprotection to photosynthetic organisms, the eye and the skin. The protection mechanisms involve both quenching of singlet oxygen and of damaging free radicals. The mechanisms for singlet oxygen quenching and protection against free radicals are quite different - indeed, under some conditions, quenching of free radicals can lead to a switch from a beneficial anti-oxidant process to damaging pro-oxidative situation. Furthermore, while skin protection involves β-carotene or lycopene from a tomato-rich diet, protection of the macula involves the hydroxyl-carotenoids (xanthophylls) zeaxanthin and lutein. Time resolved studies of singlet oxygen and free radicals and their interaction with carotenoids via pulsed laser and fast electron spectroscopy (pulse radiolysis) and the possible involvement of amino acids are discussed and used to (1) speculate on the anti- and pro-oxidative mechanisms, (2) determine the most efficient singlet oxygen quencher and (3) demonstrate the benefits to photoprotection of the eye from the xanthophylls rather than from hydrocarbon carotenoids such as β-carotene.     

Molecular Weight Dependence of Exciton Diffusion in Poly(3‐hexylthiophene)

A joint experimental and theoretical study of singlet exciton diffusion in spin‐coated poly(3‐hexylthiophene) (P3HT) films and its dependence on molecular weight is presented. The results show that exciton diffusion is fast along the co‐facial π–π aggregates of polymer chromophores and about 100 times slower in the lateral direction between aggregates. Exciton hopping between aggregates is found to show a subtle dependence on interchain coupling, aggregate size, and Boltzmann statistics. Additionally, a clear correlation is observed between the effective exciton diffusion coefficient, the degree of aggregation of chromophores, and exciton delocalization along the polymer chain, which suggests that exciton diffusion length can be enhanced by tailored synthesis and processing conditions.     

"Mitochondrial" photochemical drugs do not release toxic amounts of 1O(2) within the mitochondrial matrix space

Previously, we demonstrated that mitochondrial NAD(P)H is the primary target of singlet oxygen (1O(2)) generated by photoactivation of mitochondria-selective rhodamine derivatives. Hence, local NAD(P)H oxidation/fluorescence decrease may be used to reveal the site of intracellular 1O(2) generation. Therefore, in addition to the previously used tetramethylrhodamine methylester (TMRM), 2('),4('),5('),7(')-tetrabromorhodamine 123 bromide (TBRB) and rhodamine 123 (Rho 123), we tested here whether mitochondrial NAD(P)H of cultured hepatocytes is directly oxidized upon irradiation of different "mitochondrial" photosensitizers (Photofrin; protoporphyrin IX; Al(III) phthalocyanine chloride tetrasulfonic acid; meso-tetra(4-sulfonatophenyl)porphine dihydrochloride; Visudyne). In contrast to TMRM and Rho 123, which directly oxidized NAD(P)H upon irradiation, irradiation of intracellular TBRB and the photochemical drugs only indirectly affected mitochondrial NAD(P)H due to loss of mitochondrial integrity. In line with this result only TMRM and Rho 123 exclusively localized within the mitochondrial matrix. Due to these results it is doubtful whether real mitochondrial photosensitizers actually exist among the photochemical drugs applicable/used for photodynamic therapy.     

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.