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.

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.

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.

[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.

[Stability of sugar-binding proteins: D-galactose/D-glucose-binding protein from Escherichia coli and trehalose/maltose-binding protein from Thermococcus litoralis].

In this work we studied the structure and stability of sugar-binding proteins from mesophilic and thermophilic organisms which are of great importance for their possible use as sensing probe of biosensors aimed to glucose detection in the blood. The data obtained revealed the stabilizing effect of ligands on the structures of D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli and trehalose/maltose-binding protein from thermophilic bacterium Thermococcus litoralis. It was found that TMBP possess an increased stability as its structure remains native even under heating up to 95 degrees C.

[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.

[Physical-chemical properties of actin in different structural states. New ideas about its folding-unfolding pathways].

Results of actin folding-unfolding pathways examination and characterization of intermediate and misfolded states are summarized. Properties of microenvironments and peculiarities of location of tryptophan residues in protein are analysed in detail. This allowed to conclude that the main contribution to the bulk fluorescence of native protein is made by internal tryptophan residues Trp 340 and Trp 356, localized in hydrophobic regions, while tryptophan residues Trp 79 and Trp 86 are quenched. It has been shown that inactivated actin, previously regarded as an intermediate state between native and completely unfolded state of protein is in reality a misfolded aggregated state. The properties of actin in this state were characterized in detail. In particular, it is shown that inactivated actin is a monodisperse associate consisting of 15 monomer unit. Two earlier unknown intermediate states, which precede completely unfolding of protein macromolecule and formation of inactivated actin, were visualized. A new scheme of folding-unfolding processes was proposed. It is shown that the reason of anomalous effects, which are recorded for actin in solutions with small concentrations of GdnHCl, is a specific interaction of actin with a denaturant.

[Room temperature phosphorescence of amorphous aggregates and amyloid fibrils resulting from protein misfolding].

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid fibrils.