Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity

Beta-amyloid (Abeta) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Abeta responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Abeta aggregation still holds promise as a means to reduce Abeta neurotoxicity. In research presented here, we show that Hsp20, a novel alpha-crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Abeta amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Abeta of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Abeta fibril formation. Hsp20 was also able to significantly reduce Abeta toxicity to both SH-SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Abeta toxicity.     

Differential Congo Red staining: The effects of pH, non-aqueous solvents and the substrate

Summary Congo Red is an acid-base indicator dye. In free solution the colour and absorption characteristics of Congo Red depend not only on the pH but are also governed by the nature of the solvent environment. In tissue sections stained by Congo Red, alteration of the pH and the use of non-aqueous solvents can effect differential colouring of the tissue components. Stained sections of unmodified and chemically substituted celluloses show that differential red or blue coloration reflects the acidic or basic character of the substrate. In stained tissue sections, secondary protein structure and porosity of the substrate may also influence their colour. The effect of non-aqueous solvents is probably to modify the ionization state of the dye- substrate complex, thus altering the colour of the Congo Red. Such solvents may also change the aggregation or solvation states of the dye, with consequent modification in the colour of tissue components.     

Pinacyanol as effective probe of fibrillar beta-amyloid peptide: comparative study with Congo Red

The binding of pinacyanol (PIN), a cationic cyanine dye, to beta-amyloid fibrils (Abeta), which are associated with Alzheimer disease, was quantified by absorption spectrophotometry to measure the concentration of PIN bound to Abeta as a function of the Abeta concentration or by means of the separation of free PIN from bound PIN by centrifugation and subsequent analysis of the supernatant by visible-absorption spectrophotometry. Both methods gave equivalent results. The stoichiometry of PIN binding to Abeta was 1, and the curve representing the concentration effect of Abeta on the concentration of a dye-Abeta complex showed a biphasic curve instead of the hyperbolic curve that is characteristic of weak ligand-macromolecule interactions [e.g., as shown by Congo Red (CR)]). This and the fact that a Scatchard plot could not be fitted to the experimental data suggested that PIN binds tightly to Abeta. A comparison to the interaction of CR with Abeta led us to conclude that PIN is more sensitive than CR.     

Disaggregating effects of ethanol at low concentration on beta-poly-L-lysines

Protein aggregation is involved in a number of disorders, such as Alzheimer's disease, cystic fibrosis, and prion diseases. Such aggregates are formed by peptides in beta-conformation. The study of the processes of aggregation or its inhibition makes it necessary for the peptide to remain in a monomeric state at the beginning of aggregation assays. Using three poly-L-lysine as a model of beta-peptide, we measured the spectral changes occurring in the visible spectrum of Congo Red (CR), a diazo dye, in two solvent media, namely, an aqueous solution of ethanol 10% (v/v), and an aqueous solution of dimethyl sulfoxide (DMSO) 5% (v/v). Aggregation constants show that the presence of ethanol at low concentration produces a disaggregating effect, regardless of the degree of polymerisation of the peptide. This effect is considered to be due to the direct binding of ethanol molecules to the peptide. This binding undergoes an enhancement of the electrostatic repulsion among charged lysine chains.     

Time scale of protein aggregation dictated by liquid-liquid demixing

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.     

Induced circular dichroism as a probe of Cibacron Blue and Congo Red bound to dehydrogenases

We have investigated the circular dichroism induced in Cibacron Blue and Congo Red upon binding to several dehydrogenases to probe the conformation of the bound dyes. The circular dichroism spectra of Congo Red are quite similar when the dye is bound to lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase but has bands of opposite sign when bound to cytoplasmic malic dehydrogenase. The circular dichroism spectra of Cibacron Blue bound to these same dehydrogenases are quite different from one another. Since circular dichroism is sensitive to the conformation of bound dye, these differences argue for at least local changes in dye conformation or environment when bound to different dehydrogenases. Congo Red appears to be less sensitive to these effects than Cibacron Blue.     

A beta fibrillogenesis: kinetic parameters for fibril formation from congo red binding

Using Scatchard analysis, we have formulated as a function of time and pH the relationship between the binding of Congo red to Alzheimer's beta-amyloid and the aggregation number (i.e., monomer concentration within fibrils) as defined by nucleation-dependent self-assembly. This provides a basis on which to determine the kinetic parameters for fibril formation from the observed concentration of bound Congo red.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Self-assembly of Congo Red—A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions

The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. Two different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. Molecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr. © 1998 John Wiley & Sons, Inc. Biopoly 46: 267–281, 1998     

Characterization of methylglyoxal-modified human IgG by physicochemical methods

Human IgG is a defence protein and quite reactive to dicarbonyls. In this study, methylglyoxal-induced modification of IgG was examined by various biochemical and biophysical methods. The methylglyoxal-induced changes in IgG were monitored by UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy, 1-anilinonaphthalene-8-sulfonic acid (ANS), and thermal denaturation studies. Aggregate formation was studied by Thioflavin T (ThT), Congo red (CR) and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spectroscopic studies suggested gross changes in MGO-modified IgG. Fluorogenic AGEs appeared during modification and the MGO-modified IgG gained thermostability. The reaction produced oxidative stress in the medium because carbonyl content increased manifold and sulfhydryl groups decreased. Enhanced binding of the MGO-modified IgG by Congo red and Thioflavin T suggests crosslinking and aggregation. This was supported by SEM and TEM results.     

The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide

Alzheimer's disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Abeta). One approach for treating AD is by blocking Abeta aggregation. Activity-dependent neuroprotective protein contains a peptide, NAP that protects neurons in culture against Abeta toxicity. Here, NAP was shown to inhibit Abeta aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Abeta deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin-NAP binding to Abeta. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.     

Effect of Congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar and form microcolonies. Spontaneous variants of A. brasilense with rapid swarming are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are (a) necessary for swarming and/or spreading with the formation of microcolonies and (b) capable of interacting with Congo Red.     

一株产漆酶真菌新月弯孢霉JQH-100在染料脱色中的应用

从感染叶斑病的玉米叶片中分离、纯化得到一株高产漆酶的新月弯孢霉Curvularia lunata JQH-100菌株。液体培养Curvularia lunata JQH-100可产漆酶且活性较高,产酶高峰出现在第3天;以ABTS为底物粗酶液的最适反应温度是30℃,最适反应pH是2.8;染料脱色的研究表明,共培养体系对茜素红的脱色率达到了92.6%,对中性红和刚果红的脱色率也都在80%以上;Curvularia lunata JQH-100所产漆酶经纯化后对染料茜素红和刚果红有较高的脱色率,分别为82.1%和81.2%。研究结果显示Curvularia lunata JQH-100在染料废水处理中有较大应用潜力。     

Amphotericin B binds to amyloid fibrils and delays their formation: a therapeutic mechanism?

The membrane-active antifungal agent amphotericin B (AmB) is one of the few agents shown to slow the course of prion diseases in animals. Congo Red and other small molecules have been reported to directly inhibit amyloidogenesis in both prion and Alzheimer peptide model systems via specific binding. We propose that it is possible that AmB may act similarly to physically prevent conversion of the largely alpha-helical prion protein (PrP) to the pathological beta-sheet aggregate protease-resistant isoform (PrP(res)) in prion disease and by analogy prevent fibrillization in amyloid diseases. To assess whether AmB is capable of binding specifically to amyloid fibrils as does Congo Red, we have used the insulin fibril and Abeta 25-35 amyloid model fibril system. We find that AmB does bind strongly to both insulin (K(d) = 1.1 microM) and Abeta 25-35 amyloid (K(d) = 6.4 microM) fibrils but not to native insulin. Binding is characterized by a red-shifted AmB spectrum indicative of a more hydrophobic environment. Thus AmB seems to have a complementary face for amyloid fibrils but not the native protein. In addition, AmB interacts specifically with Congo Red, a known fibril-binding agent. In kinetic fibril formation studies, AmB was able to significantly kinetically delay the formation of Abeta 25-35 fibrils at pH 7.4 but not insulin fibrils at pH 2.     

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.     

A prionogenic peptide derived from Sup35 can force the whole GST fusion protein to show amyloid characteristics

A prion determining 7-mer peptide derived from Sup35 was fused to glutathione S transferase (GST). The fusion protein was successfully overexpressed in Escherichia coli, and purified by employing affinity chromatography. Upon incubation, it showed substantial aggregation suggesting the formation of amyloid-like fibrils. Congo Red binding strongly suggested that the fusion protein formed amyloid-like fibrils. By considering the steric hindrance of GST, the beta-sheet formation should be in the anti-parallel fashion.     

Demonstration of amyloid with Mesitol WLS-Congo Red: Application of a textile auxiliary to histochemistry

Summary Previous histochemical investigations demonstrated similarities in the binding of Congo Red and other direct cotton dyes by amyloid and cellulose. It seemed threfore of interest to determine whether or not the cellulose-like reactivity of amyloid extends also to dye solutions containing an anionic reserving agent. These reagents are used in the dyeing of wool-cellulose (Halbwolle) fabrics to prevent binding of direct cotton dyes by proteins. Mesitol WLS-Congo Red solutions stained amyloid selectively; other tissue structures, except some hyaline deposits in arterioles, remained unstained. The cause of this non-specific reaction could not be determined with certainty. Therefore, the alkaline Congo Red method is recommended for histochemical identification of amyloid. However, the Mesitol WLS-Congo Red technic was very useful for demonstration of amyloid after prolonged storage of tissues in formalin; amyloid in such material showed little or no reactivity with the alkaline Congo Red or the Sirius dye methods. This pilot study indicates that anionic reserving agents can be effectively employed under conditions of histochemical technics.     

In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells

Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T_m at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO₄ ⁻≫Cl⁻>H₂PO₄ ⁻, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Oligomerization and transglutaminase cross-linking of the cystatin CRES in the mouse epididymal lumen: potential mechanism of extracellular quality control

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.