Structural determinants of odorant-binding proteins affecting their ability to form amyloid fibrils.

The formation of amyloid fibrils is associated with many severe pathologies as well as the execution of essential physiological functions by proteins. Despite the diversity, all amyloids share a similar morphology and consist of stacked ß-strands, suggesting high amyloidogenicity of native proteins enriched with ß-structure. Such proteins include those with a ß-barrel-like structure with ß-strands arranged into a cylindrical ß-sheet. However, the mechanisms responsible for destabilization of the native state and triggering fibrillogenesis have not thoroughly explored yet. Here we analyze the structural determinants of fibrillogenesis in proteins with ß-barrel structures on the example of odorant-binding protein (OBP), whose amyloidogenicity was recently demonstrated in vitro. We reveal a crucial role in the fibrillogenesis of OBPs for the "open" conformation of the molecule. This conformation is achieved by disrupting the interaction between the ß-barrel and the C-terminus of protein monomers or dimers, which exposes "sticky" amyloidogenic sites for interaction. The data suggest that the "open" conformation of OBPs can be induced by destabilizing the native ß-barrel structure through the disruption of: 1) intramolecular disulfide cross-linking and non-covalent contacts between the C-terminal fragment and ß-barrel in the protein's monomeric form, or 2) intermolecular contacts involved in domain swapping in the protein's dimeric form.

Mammalian odorant-binding proteins are prone to form amorphous aggregates and amyloid fibrils.

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's ß-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions. Changes in environmental parameters (increased temperature and pH) favored the formation of larger and dense supramolecular complexes that significantly reduce the binding of ligands by bOBP. The ability of bOBP to form fibrillar aggregates with the properties of amyloids, including high cytotoxicity, was revealed at sample stirring (even at physiological temperature and pH), at medium acidification or pre-solubilization with hexafluoroisopropanol. Fibrillogenesis of bOBP was initiated by the dissociation of the protein's supramolecular complexes into monomers and the destabilization of the protein's ß-barrels without a significant destruction of its native ß-strands.

Amyloid Fibrils of Pisum sativum L. Vicilin Inhibit Pathological Aggregation of Mammalian Proteins.

Although incurable pathologies associated with the formation of highly ordered fibrillar protein aggregates called amyloids have been known for about two centuries, functional roles of amyloids have been studied for only two decades. Recently, we identified functional amyloids in plants. These amyloids formed using garden pea Pisum sativum L. storage globulin and vicilin, accumulated during the seed maturation and resisted treatment with gastric enzymes and canning. Thus, vicilin amyloids ingested with food could interact with mammalian proteins. In this work, we analyzed the effects of vicilin amyloids on the fibril formation of proteins that form pathological amyloids. We found that vicilin amyloids inhibit the fibrillogenesis of these proteins. In particular, vicilin amyloids decrease the number and length of lysozyme amyloid fibrils; the length and width of ß-2-microglobulin fibrils; the number, length and the degree of clustering of ß-amyloid fibrils; and, finally, they change the structure and decrease the length of insulin fibrils. Such drastic influences of vicilin amyloids on the pathological amyloids' formation cause the alteration of their toxicity for mammalian cells, which decreases for all tested amyloids with the exception of insulin. Taken together, our study, for the first time, demonstrates the anti-amyloid effect of vicilin fibrils and suggests the mechanisms underlying this phenomenon.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aß-peptide (Aß42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

sfGFP throws light on the early stages of ß-barrel amyloidogenesis.

The accumulation of ß-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native ß-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic ß-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate "native-like" state preserving the integrity of the sfGFP ß-barrel scaffold despite the partial distortion of the three ß-strands closing it. The interaction between the "melted" regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the ß-barrel opens and some of the native α-helices and disordered regions refold into non-native ß-strands, which, along with native ß-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other ß-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis.

New Evidence on a Distinction between Aß40 and Aß42 Amyloids: Thioflavin T Binding Modes, Clustering Tendency, Degradation Resistance, and Cross-Seeding.

The relative abundance of two main Abeta-peptide types with different lengths, Aß40 and Aß42, determines the severity of the Alzheimer's disease progression. However, the factors responsible for different behavior patterns of these peptides in the amyloidogenesis process remain unknown. In this comprehensive study, new evidence on Aß40 and Aß42 amyloid polymorphism was obtained using a wide range of experimental approaches, including custom-designed approaches. We have for the first time determined the number of modes of thioflavin T (ThT) binding to Aß40 and Aß42 fibrils and their binding parameters using a specially developed approach based on the use of equilibrium microdialysis, which makes it possible to distinguish between the concentration of the injected dye and the concentration of dye bound to fibrils. The binding sites of one of these modes located at the junction of adjacent fibrillar filaments were predicted by molecular modeling techniques. We assumed that the sites of the additional mode of ThT-Aß42 amyloid binding observed experimentally (which are not found in the case of Aß40 fibrils) are localized in amyloid clots, and the number of these sites could be used for estimation of the level of fiber clustering. We have shown the high tendency of Aß42 fibers to form large clots compared to Aß40 fibrils. It is probable that this largely determines the high resistance of Aß42 amyloids to destabilizing effects (denaturants, ionic detergents, ultrasonication) and their explicit cytotoxic effect, which we have shown. Remarkably, cross-seeding of Aß40 fibrillogenesis using the preformed Aß42 fibrils changes the morphology and increases the stability and cytotoxicity of Aß40 fibrils. The differences in the tendency to cluster and resistance to external factors of Aß40 and Aß42 fibrils revealed here may be related to the distinct role they play in the deposition of amyloids and, therefore, differences in pathogenicity in Alzheimer's disease.

Yeast red pigment, protein aggregates, and amyloidoses: a review.

Estimating the amyloid level in yeast Saccharomyces, we found out that the red pigment (product of polymerization of aminoimidazole ribotide) accumulating in ade1 and ade2 mutants leads to drop of the amyloid content. We demonstrated in vitro that fibrils of several proteins grown in the presence of the red pigment stop formation at the protofibril stage and form stable aggregates due to coalescence. Also, the red pigment inhibits reactive oxygen species accumulation in cells. This observation suggests that red pigment is involved in oxidative stress response. We developed an approach to identify the proteins whose aggregation state depends on prion (amyloid) or red pigment presence. These sets of proteins overlap and in both cases involve many different chaperones. Red pigment binds amyloids and is supposed to prevent chaperone-mediated prion propagation. An original yeast-Drosophila model was offered to estimate the red pigment effect on human proteins involved in neurodegeneration. As yeast cells are a natural feed of Drosophila, we could compare the data on transgenic flies fed on red and white yeast cells. Red pigment inhibits aggregation of human Amyloid beta and α-synuclein expressed in yeast cells. In the brain of transgenic flies, the red pigment diminishes amyloid beta level and the area of neurodegeneration. An improvement in memory and viability accompanied these changes. In transgenic flies expressing human α-synuclein, the pigment leads to a decreased death rate of dopaminergic neurons and improves mobility. The obtained results demonstrate yeast red pigment potential for the treatment of neurodegenerative diseases.

New findings on GFP-like protein application as fluorescent tags: Fibrillogenesis, oligomerization, and amorphous aggregation.

Green fluorescent proteins (GFP) are commonly used as fluorescent tags and biosensors in cell biology and medicine. However, the propensity of GFP-like proteins to aggregate and the consequence of intermolecular interaction for their application have not been thoroughly examined. In this work, alternative aggregation pathways of superfolder green fluorescent protein (sfGFP) were demonstrated using a spectroscopic and microscopic study of the samples prepared by equilibrium microdialysis. Besides oligomerization of native monomers, we showed for the first time the condition-specific formation by sfGFP of either amyloid fibrils (at increased temperature or acidity) or amorphous aggregates (at physiological conditions). Both types of sfGFP aggregates had lost green fluorescence and were toxic to cells. Thus, when using GFP-like proteins as fluorescent tags, one should take into account their high ability to form aggregates with lost unique visible fluorescence in the cellular environment, which affects cell viability. Moreover, the results of this work cast doubt on the correctness of the data on the fibrillogenesis of various amyloidogenic proteins obtained using their fusion with GFP-like proteins.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Yeast red pigment modifies Amyloid beta growth in Alzheimer disease models in both Saccharomyces cerevisiae and Drosophila melanogaster.

The effect of yeast red pigment on amyloid-ß (Aß) aggregation and fibril growth was studied in yeasts, fruit flies and in vitro. Yeast strains accumulating red pigment (red strains) contained less amyloid and had better survival rates compared to isogenic strains without red pigment accumulation (white strains). Confocal and fluorescent microscopy was used to visualise fluorescent Aß-GFP aggregates. Yeast cells containing less red pigment had more Aß-GFP aggregates despite the lower level of overall GFP fluorescence. Western blot analysis with anti-GFP, anti-Aß and A11 antibodies also revealed that red cells contained a considerably lower amount of Aß GFP aggregates as compared to white cells. Similar results were obtained with exogenous red pigment that was able to penetrate yeast cells. In vitro experiments with thioflavine and TEM showed that red pigment effectively decreased Aß fibril growth. Transgenic flies expressing Aß were cultivated on medium containing red and white isogenic yeast strains. Flies cultivated on red strains had a significant decrease in Aß accumulation levels and brain neurodegeneration. They also demonstrated better memory and learning indexes and higher locomotor ability.

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition.

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane's phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.

The effect of red pigment on the amyloidization of yeast proteins.

The intensity of amyloid-bound thioflavine T fluorescence was studied in crude lysates of yeast strains carrying mutations in the ADE1 or ADE2 genes and accumulating the red pigment (a result of polymerization of aminoimidazoleribotide), and in white isogenic strains--either adenine prototrophs or carrying mutations at the first stages of purine biosynthesis. We found that the red pigment leads to a drop of amyloid content. This result, along with the data on separation of protein polymers of white and red strains in PAGE, suggests that the red pigment inhibits amyloid fibril formation. The differences in transmission of the thioflavine T fluorescence pattern by cytoduction and in blot-hybridization of pellet proteins of red and white [PSI(+) ] strains with Sup35p antibodies confirmed this conclusion. Purified red pigment treatment also led to a decrease of fluorescence intensity of thioflavine T bound to insulin fibrils and to yeast pellet protein aggregates from [PSI(+) ] strains. This suggests red pigment interaction with amyloid fibrils. Comparison of pellet proteins from red and white isogenic strains separated by 2D-electrophoresis followed by MALDI analysis has allowed us to identify 48 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, closely corresponding to prion-dependent proteins that we characterized previously. Also present were some proteins involved in stress response and proteolysis. We suppose that the red pigment acts by blocking certain sites on amyloid fibrils that, in some cases, can lead in vivo to interfere with their contacts with chaperones and the generation of prion seeds.

Prion-associated proteins in yeast: comparative analysis of isogenic [PSI(+)] and [psi(-)] strains.

A large group of prion-associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two-dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI(+)] and [psi(-)] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion-containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion-associated, but we cannot exclude the possibility that several proteins may propagate as prions.

Bud selection and apoptosis-like degradation of nuclei in yeast heterokaryons: a KAR1 effect.

It has been shown that defects in cell fusion during mating can trigger programmed cell death in the yeast Saccharomyces cerevisiae. We wished to test whether defects in nuclear migration during cell fusion have the same effect. A partial pedigree analysis of nine kar1 x KAR1 crosses of two different types (four alpha KAR1 x a kar1 and five alpha kar1 x a KAR1 crosses) was carried out, and quantitative estimates of the frequencies of different mother/daughter (m/d) classes were obtained. The kar1 mutation affects nuclear congression and delays nuclear fusion. In each cross tested, the nucleus that entered the first bud tended to be the one contributed by the cell that carried the wild-type allele of KAR1. If budding was delayed by nutrient limitation, the kar1 nucleus could be rescued, indicating that the primary effect of the kar1 mutation is that it slows spindle action. Many m/d classes appear as a result of the degradation of one of the nuclei in the heterokaryon. Loss of nuclei in heterokaryons was accompanied by an accumulation of reactive oxygen species (ROS), and by abnormalities in nuclear structure revealed by TUNEL (terminal transferase-mediated dUTP nick end-labeling) analysis, DAPI staining and by histone-GFP fluorescence patterns which suggested an apoptosis-like process. Often only one nucleus was degraded, and ROS accumulation was restricted to one half of the zygote. We therefore suggest that the data obtained can be explained by apoptosis-like death of a half-cell (cell body).

The impact of manipulations with cytoplasmically inherited factors on nuclear transmission and degradation in yeast heterokaryons.

Heterokaryotic zygotes in yeast provide a unique possibility to study the survival and transmission of two genetically diverse nuclei in one cell. Using partial pedigree analysis, we show that various treatments used to change cytoplasmic hereditary determinants can essentially affect nuclear transmission in yeast heterokaryons. This includes choice of nucleus to enter the first bud and incidence of various classes of mother/daughter pairs demonstrating nuclear degradation patterns in heterokaryotic zygotes. These treatments include guanidine hydrochloride, a prion-curing agent, ethidium bromide, an agent causing elimination of mitochondrial DNA, and cytoplasm replacement by cytoduction, which leads to mtDNA replacement and transfer of some other cytoplasmically inherited determinants. The genetic and cytological evidence obtained favors prion involvement in nuclear transmission and suggests apoptotic features in nuclear degradation in yeast heterokaryotic zygotes.