Alexei V. Feofanov

Chromophore environment provides clue to "kindling fluorescent protein" riddle.

asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemoneAnemonia sulcata, becomes fluorescent (“kindles”) upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be “quenched” instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr66 in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

Latarcins, Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) That Exemplify Biomolecular Diversity

Seven novel short linear antimicrobial and cytolytic peptides named latarcins were purified from the venom of the spider Lachesana tarabaevi. These peptides were found to produce lytic effects on cells of diverse origin (Gram-positive and Gram-negative bacteria, erythrocytes, and yeast) at micromolar concentrations. In addition, five novel peptides that share considerable structural similarity with the purified latarcins were predicted from the L. tarabaevi venom gland expressed sequence tag data base. Latarcins were shown to adopt amphipathic α-helical structure in membrane-mimicking environment by CD spectroscopy. Planar lipid bilayer studies indicated that the general mode of action was scaled membrane destabilization at the physiological membrane potential consistent with the “carpet-like” model. Latarcins represent seven new structural groups of lytic peptides and share little homology with other known peptide sequences. For every latarcin, a precursor protein sequence was identified. On the basis of structural features, latarcin precursors were split into three groups: simple precursors with a conventional prepropeptide structure; binary precursors with a typical modular organization; and complex precursors, which were suggested to be cleaved into mature chains of two different types.

Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage

Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.

Confocal Raman Microspectroscopy and Imaging Study of Theraphthal in Living Cancer Cells

Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing effect on tumor cells is achieved by production of free radicals in the course of accelerated oxidation of ascorbate by dioxygen in the presence of transition metal complexes. Further progress in the development of binary catalytic systems (BCSs) requires a special method for their investigation in cells and tissues, because neither component of BCSs fluoresces. Here a resonance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retention of theraphthal (TP), a catalyst of the advanced TP/ascorbate BCS, were investigated in A549 cells with the RR CSI technique. The cellular analysis was complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca(2+) cations and decreasing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were discriminated, namely, monomeric TP in polar environment, TP bound to proteins, and aggregated TP. Their cytoplasmic and nuclear distributions were mapped at different stages of uptake and efflux. By introducing organelle-selective fluorescent probes into drug-treated cells and measuring intracellular localization of both the probe and the drug, compartmentation of TP was revealed. Cell growth suppression by the TP/ascorbate system was measured, and probable molecular and organelle targets of radical damage were characterized.

Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding

Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)‐binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3′‐azido‐3′‐deoxythymidine (a ligand occupying IIIA structural sub‐domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA‐bound CPT may be replaced by warfarin, occupying IIA structural sub‐domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT‐binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT‐induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp‐residue to hydrophilic environment. These data suggest that CPT is bound in the region of inter‐domain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

Quantitative confocal spectral imaging analysis of mitoxantrone within living K562 cells: intracellular accumulation and distribution of monomers, aggregates, naphtoquinoxaline metabolite, and drug-target complexes.

Confocal spectral imaging (CSI) technique was used for quantitative analysis of the uptake, subcellular localization, and characteristics of localized binding and retention of anticancer agent mitoxantrone (MITOX) within human K562 erythroleukemia cells. The CSI technique enables identification of the state and interactions of the drug within the living cells. Utilizing this unique property of the method, intracellular distributions were examined for monomeric MITOX in polar environment, MITOX bound with hydrophobic cellular structures, naphthoquinoxaline metabolite, and nucleic acid-related complexes of MITOX. The features revealed were compared for the cells treated with 2 microM or 10 microM of MITOX for 1 h and correlated to the known data on antitumor action of the drug. MITOX was found to exhibit high tendency to self-aggregation within intracellular media. The aggregates are concluded to be a determinant of long-term intracellular retention of the drug and a source of persistent intracellular binding of MITOX. Considerable penetration of MITOX in the hydrophobic cytoskeleton structures as well as growing accumulation of MITOX bound to nucleic acids within the nucleus were found to occur in the cells treated with a high concentration of the drug. These effects may be among the factors stimulating and/or accompanying high-dose mitoxantrone-induced programmed cell death or apoptosis.

Synthesis, Structure, Spectroscopic Studies, and Complexation of Novel Crown Ether Butadienyl Dyes

Butadienyl dyes of the benzothiazole series with various fragments of benzocrown ethers 1a-c were synthesized for the first time. The structures and spectral properties of crown-containing butadien ...

Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis.

Studying mechanisms of drug antitumor action is complicated by the lack of noninvasive methods enabling direct monitoring of the state and interactions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming this problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, 8-bis-[([2-(2-hydroxyethyl)-amino]ethyl)amino]-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spectra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target complexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affecting drug fluorescence. MITOX exposed to aqueous intracellular environment, MITOX bound to hydrophobic cellular structures, complexes of MITOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These states and complexes are known to be immediately related to the antitumor action of the drug. The results obtained present a basis for the subsequent quantitative analysis of concentration and time-dependent accumulation of free and bound MITOX within different compartments of living cancer cells.

Near‐infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15‐N‐(3′‐Hydroxypropyl)Cycloimide Chlorin p6¶

The 13,15‐N‐(3′‐hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell‐killing activity. It is 43‐, 61‐ and 110‐fold more active than chlorin p6, 3‐formyl‐3‐devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002–0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 ± 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3‐ to 4‐fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 ± 5 min. The time for 50% release of CIC from the cells is 60 ± 10 min. A 10‐fold decrease in CIC intracellular penetration at 22°C proves that temperature‐sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2–1.6 μM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic‐type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near‐IR photosensitizer.

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE*

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.