Microfluidic Chip as a Tool for Effective In Vitro Evaluation of Cyclophosphamide Prodrug Toxicity.

Most drugs are metabolized in the liver, which can lead to their activation or inactivation with a change in the parent compound pharmacology, as well as liver damage by active metabolites. Preclinical animal studies of drug safety do not always predict its effect on humans due to species specificity. Thus, for the rapid drug screening, and especially prodrugs, an in vitro system is required that allows predicting xenobiotic cytotoxicity with consideration of their metabolism in liver cells. The use of a microfluidic chip (BioClinicum) made it possible to cultivate a 2D culture of human HaCaT keratinocytes with spheroids of human hepatoma HepaRG cells. After incubation in a specially selected universal serum-free medium containing 3.8 mM cyclophosphamide, pronounced death of HaCaT cells was observed in comparison with culturing in the absence of liver cells.

Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity.

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient's body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins' effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils.

Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

Denaturant effect on amyloid fibrils: Declasterization, depolymerization, denaturation and reassembly.

Accumulation of amyloid fibrils in organism accompanies many serious diseases, such as Alzheimer's and Parkinson's diseases, diabetes, prion diseases, etc. It is generally accepted that amyloids are highly resistant to degradation, which complicates their elimination in vivo and is one of the reasons for their pathogenicity. However, using a wide range of physicochemical approaches and specially elaborated method for the tested samples preparation by equilibrium microdialysis technique, it is proved that the stability of amyloids is greatly exaggerated. It turned out that amyloid fibrils formed from at least two amyloidogenic proteins, one of which is a model object for fibrils studying and the second is the cause of hemodialysis amyloidosis in an acute renal failure, are less stable than monomeric proteins. A mechanism of the degradation/reassembly of amyloid fibrils was proposed. It was shown that amyloid «seed¼ is a factor affecting not only the rate of the fibrils formation, but also their structure. Obtained results are a step towards identifying effects that can lead to degradation of amyloids and their clearance without adverse influence on the functionally active state of the protein or to change the structure and, as a result, the pathogenicity of these protein aggregates.

Structural Analogue of Thioflavin T, DMASEBT, as a Tool for Amyloid Fibrils Study.

Fluorescent dye trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-benzothiazolium perchlorate (DMASEBT) is a relatively recently synthesized probe for detection of amyloid fibrils accumulating in the organs and tissues of patients with a wide range of serious incurable diseases. DMASEBT was developed as an alternative of its widely used analogue thioflavin T (ThT), which is the "gold standard" for the amyloid fibrils study. Our results show the similarity of both dyes binding to amyloid fibrils and allow one to propose a mechanism of such probes interaction with some types of the fibrils. At the same time, DMASEBT has a significant advantage, namely, improved photophysical properties compared with ThT, which allows for the detection of DMASEBT-stained amyloid fibrils in the spectral region of the "transparency window of biological tissues". The ability of the dye to penetrate into the cells was shown to open the prospect of this dye's use for amyloid fibrils bioimaging and biosensing in vivo. Furthermore, it was proven that DMASEBT can be used not only as a test for amyloid fibrils formation but also for the comparative study of the fibrils structure (both their fibers and bunches), which in turn may underlie the variability of amyloidosis and affect the cytotoxicity of these protein aggregates.

Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis.

The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effective test for protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of ThT-AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis, we proposed a new experimental approach for examination of ThT-AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed us to prove 1/1 ThT/AChE binding stoichiometry. The increase of ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in ThT-AChE interaction, as observed for some amyloid fibrils.

[Protein Folding and Stability in the Presence of Osmolytes].

Osmolytes are molecules with the function among others to align hydrostatic pressure between intracellular and extracellular spaces. Accumulation of osmolytes occurs in the cell in response to stress caused by pressure change, change in temperature, pH, and concentration of inorganic salts. Osmolytes can prevent native proteins denaturation and promote folding of unfolding proteins. Investigation of the osmolytes effect on these processes is essential for understanding the mechanisms of folding and functioning of proteins in vivo. A score of works, devoted to the effect of osmolytes on proteins, are not always consistent with each other. In this review an attempt was made to systemize available array of data on the subject and consider the problem of folding and stability of proteins in solutions in the presence of osmolytes from the single viewpoint.

[GLOBULAR ACTIN IS THE PARTIALLY INTRINSICALLY DISORDERED PROTEIN WITH QUASI-STATIONARY STRUCTURE].

It is shown that the native globular actin (G-actin) is the thermodynamically unstable (quasi-stationary) form of the protein. This state is stabilized by Mg2+ (in vitro replaced by Ca2+). In vivo this state occurs as a result of complex energy-consuming post-translational folding processes including chaperone Hsp70, prefoldin and CCT complex, providing the formation of the native structure stabilized by Ca2+ and ATP. Structures formed by actin polypeptide chain constantly form complexes with their partners (chaperones Hsp70, prefoldin and chaperonin CCT in folding process, with an Mg ion and ATP in the native state, with numerous actin-binding proteins during the formation and functioning of the cell cytoskeleton, with myosin and other proteins of the muscle contraction in the muscle cells). Actin denaturation is accompanied by self-association of molecules, so the inactivated actin is the thermodynamically stable compact structure consisting of 14-16 protein molecules. Apparently, proteins with quasi-stationary native state are widespread in nature. The emergence of these states is energy-consuming and is conjugated with the inability of the polypeptide chain to form the native compact structure without assistants (complex machinery of protein folding in the cell) and without interaction with their natural partners, in particular with metal ions.

[Knotted proteins].

For a long time the presence of knots in a protein structure was not admitted. However, the existence of proteins with various types of knots has now been proven. The functional significance of knotted topology remains unclear despite numerous assumptions. Studing the structure of knots in proteins and their impact on the acquisition of native structure of proteins is important for the understanding of protein folding as a whole. We review the types of knots in the proteins discovered to date, including trefoil knot, figure-of-eight knot, and more complex knots with 5 and 6 crossings of polypeptide chain. We survey the folding of knotted proteins as well.

[Physical-chemical properties of the mutant (protein) form of D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN].

The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system.

[Investigation of the kinetics of insulin amyloid fibrils formation].

Investigation of the structure of ordered protein aggregates--amyloid fibrils, the influence of the native structure of the protein and the environment on the process of fibrillation is currently the subject of intensive research. The present work is devoted to the study of the kinetics of insulin amyloid fibrils formation at low pH values (which are produced at many stages of the isolation and purification of the protein) using a fluorescent probe thioflavin T (ThT). It has been shown that the increase of fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which 3 areas can be distinguished: the lag phase, the growth and the plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), we have found its reduction with stirring the solution and seeding. Data obtained using electron microscopy showed that the formed fibrils are long, linear filament having a diameter of -20 nm. With increasing incubation time fibril diameter did not change while their length increases to 2-3 µm, which was accompanied by a significant increase in the number of aggregates of fibrils. All the experimental data shows that, regardless of the kinetics of the formation of amyloid fibrils, their properties after the fibrillation process are identical. The results of this work together with the previously studies of insulin amyloid fibrils might be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.

[Comparative structural and functional characteristics of different forms of Saccharomyces cerevisiae red pigment and its synthetic analogue].

Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(beta-D-ribofuranosyl)-5-aminoimadazole), isolated from adel 1 mutant cells of Saccharomyces cerevisiae, its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimadazole in vitro) has been obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. The entire compounds studied were able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in presence of these compounds--they were merged into conglomerates that were more stable and resistant to the effects of ultrasound in comparison with insulin aggregates grown without pigments. We speculate that all these compounds can cause coalescence of fibrils, partially block their loose ends and, thereby, inhibit the attachment of new monomers to growing fibrils.

[The effect of red pigment on amyloidization of yeast proteins].

Amyloid bound thioflavine T fluorescence was studied in the lysates of yeast strains carrying mutations in genes ADE1 or ADE2 and accumulating red pigment, a result of polymerization of aminoimidazoleribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in the case of cells grown in media containing high concentration of adenine (100 mg/l) that blocks accumulation of red pigment. Blocks at first stages of purine biosynthesis de novo also impede red pigment and lead to the same effect on thioflavine fluorescence. At the same time induction of mutations in genes ADE1 or ADE2 in originally white prototrophic strains leads to considerable drop of fluorescence. A fraction of protein polymers was studied by agarose gel electrophoresis and this permitted to conclude that lowering of fluorescence intensity is indeed connected with the decrease of amyloid amount in cells accumulating red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with Thioflavine T. 2D-electrophoretic comparison of pellet proteins of red and white isogenic strains, followed by MALDI, allowed identification of 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins, involved in glucose metabolism, closely corresponding to prion-dependent proteins characterized in our previous work. We suppose that, binding amyloid fibrils, red pigment hinders formation of prion aggregates and also, blocking fibril contact with chaperones, impedes prion propagation.

[Comparison of crude lysate pellets of isogenic strains of yeast with different prion composition: identification of a set of prion-associated proteins].

A new approach: comparative analysis of proteins of the pellets of crude cell lysates of isogenic strains of Saccharomyces cerevisiae differing by their prion composition permitted to identify a large group of prion-associated proteins in yeast cells. 2D-electrophoresis followed by MALDI-analysis of a recipient [psi-] strain and of [PSI+] cytoductant led to identification of 35 proteins whose aggregation state responded to a shift of prion(s) content. Approximately half of these proteins belonged to functional groups of chaperones and enzyme involved in glucose metabolism. Notable were also proteins involved in translation, in oxidative stress response and in protein degradation. The data obtained are compared with the results of other groups who used other approaches to detecting proteins involved in prion aggregates.

[Thioflavin T interaction with amyloid fibrils as an instrument for their studying].

Benzthiazole dye thioflavin T (ThT) is widely used to study the formation and structure of amyloid fibrils. Nevertheless, till now there is no common opinion concerning molecular mechanisms of ThT binding to amyloid fibrils and the reasons of dramatic increase in its fluorescence quantum yield on incorporation into amyloid fibrils. Our data prove that ThT molecules incorporate in the amyloid fibrils in the monomeric form and there is no ground to suppose the formation of ThT dimers, eximers, or micells. It was shown that the increase in the quantum yield of ThT incorporated in amyloid fibrils was caused by restriction of benzthiazole and aminobenzene rings torsion fluctuations relative to each other. The use of equilibrium microdialysis allowed determining the absorption spectrum, the number of binding modes of ThT with insulin amyloid fibrils and for each mode determining the binding constants and the number of binding sites for each mode.

[Native globular and native partially or completely disordered proteins. Folding, supramolecular complex formation and aggregation].

Recently it became evident that proteins can perform their function not only in globular state but also in partially or completely disordered state. The majority of globular proteins are enzymes which function is strictly determined. Regulation and signaling proteins participating in interconnection with variety of partners must have much more lability, and macromolecules of such proteins are mainly in partially or completely disordered state. The aim of this work was to describe from the unified viewpoint in the frame of energy landscape model the existence of native globular, native partially or completely disordered proteins, formation of intermolecular complexes with various partners, formation of amorphous aggregates and amyloid fibrils. Compact globular proteins are formed if polypeptide chain provides strong intramolecular interconnections. The ability of polypeptide chain to fold in a compact globule depends on the relation of hydrophobic and charged aminoacids in its composition. Many partially or completely disordered proteins can form compact structure in complexes with their partners, which are composed by intermolecular interactions of polypeptide chains of protein and its partner. Intermolecular interaction of proteins can lead to formation associates, amorphous aggregates, amyloid and amyloid-like fibrils. The requisite condition of such contact formation is the availability of hydrophobic clusters of polypeptide chain exposed to the solution. That is why aggregation of partially or completely disordered proteins is more favorable in comparison with globular proteins.

[Estimating of changes in the amyloid and prion content of yeast cells].

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.

[Fluorescent proteins: physical-chemical properties and application in cell biology].

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

[Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris].

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.

[Structural dynamics, stability and folding of proteins].

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.