Hen Egg White Lysozyme Fibrillation Research Articles

Solubility and Supersaturation-Dependent Protein Misfolding Revealed by Ultrasonication

Although alcohols are useful cosolvents for producing amyloid fibrils, the underlying mechanism of alcohol-dependent fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 12-16%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase in fibrillation above 60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results indicate that although fibrillation is determined by solubility, supersaturation prevents conformational transitions and ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent protein misfolding funnel useful for examining amyloidogenicity. This misfolding funnel will apply to fibrillation under physiological conditions where biological environments play important roles in decreasing the solubility.

Binding of hen egg white lysozyme fibrils with nucleic acids

Non proteinaceous substances are found to be associated with toxic protein aggregates commonly known as fibrils. Hen egg white lysozyme (HEWL) is able to form fibrillar species under various conditions. Here for the first time we report concentration dependent binding affinities of preformed HEWL fibrils towards DNA and RNA at physiological pH (pH 7.4). We have found that HEWL fibrils bind with DNA and RNA that is distinctly different when compared to native HEWL. The association constant (Ka) of native HEWL and ct-DNA at pH 7.4 is 6.8×105M−1. We have also investigated the conformational alterations of DNA that occur on binding with HEWL fibrils. Our study has demonstrated dominant electrostatic interactions between oppositely charged polyelectrolytes which accounts for the binding of nucleic acids with fibrils. The affinity between the moieties could lead to disruption in the functions of cellular components that might be attributed to the toxicity of the aggregates formed in vivo.

Curcumin's pre-incubation temperature affects its inhibitory potency toward amyloid fibrillation and fibril-induced cytotoxicity of lysozyme

BackgroundMore than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL). MethodsThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability. Results and conclusionThe addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation. General significanceWe believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Mitochondrial membrane permeabilization upon interaction with lysozyme fibrillation products: Role of mitochondrial heterogeneity

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders, although the relative degree of this functional impairment and the mechanism of selective vulnerability in different regions of the brain related to various neurological diseases are not completely understood. In a recent study, we reported on brain mitochondrial membrane permeabilization upon interaction with hen egg white lysozyme (HEWL) protofibrils, and came to the conclusion that mitochondrial heterogeneity could offer an explanation for some of our observations. Accordingly, the first part of the present investigation was devoted on studies involving interaction of HEWL fibrillation products with mitochondria isolated from various areas of the brain, known to be affected in a number of well-characterized neurodegenerative conditions. This was followed by looking at heart and liver mitochondria. Membrane permeabilization was investigated by monitoring release of mitochondrial enzymes. Mitochondria isolated from cortex and hippocampus showed greater sensitivities than those prepared from substantia nigra. Results clearly demonstrate heterogeneity in brain mitochondria together with a higher resistance to permeabilization in these organelles in comparison with those isolated from liver and heart. Calcium and spermine were found to be more effective in preventing permeabilization in brain mitochondria, as compared to liver and heart. The structure–function aspects and physiological significance of the observations in relation to differences in composition, biophysical nature and morphological properties of mitochondria are discussed. It is argued that studies on heterogeneity in cellular membranes, the primary targets of toxic protofibrils, may provide important insights into mechanism of toxicity, with clinical and pathological manifestations.

Effects of copolypeptides on amyloid fibrillation of hen egg‐white lysozyme

The fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of simple, unstructured D,L-lysine-co-glycine (D,L-Lys-co-gly) and D,L-lysine-co-L-phenylalanine (D,L-Lys-co-Phe) copolypeptides was studied by using a variety of analytical techniques. The attenuating and decelerating effects on fibrillation are significantly dependent on the polypeptide concentration and the composition ratios in the polypeptide chain. Interestingly, D,L-Lys-co-gly and D,L-Lys-co-Phe copolypeptides with the same composition ratio have comparable attenuating effects on fibrillation. The copolypeptide with highest molar fraction of glycine residue exhibits the strongest suppression of HEWL fibrillation. The copolypeptide has the highest hydrophobic interacting capacity due to the more molar ratio of apolar monomer in the polymer backbone. The major driving forces for the association of HEWL and copolypeptides are likely to be hydrogen bonding and hydrophobic interactions, and these interactions reduce the concentration of free protein in solution available to proceed to fibrillation, leading to the increase of lag time and attenuation of fibrillation. The results of this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and the amyloid proteins.

Rottlerin dissolves pre-formed protein amyloid: A study on hen egg white lysozyme

BackgroundDeposition of protein fibrillar aggregates called amyloids in the tissue, is the principal cause of several degenerative diseases. Here, we have shown the disaggregation potential of rottlerin towards hen egg white lysozyme (HEWL) fibrils formed under alkaline conditions (pH-12.2). MethodsSeveral biophysical methods like Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and fluorescence emission spectra were used for the study. Results and conclusionRottlerin exhibited instantaneous disaggregation effect on HEWL fibrils as monitored by Thioflavin T assay, anisotropy study and AFM imaging. Further we have monitored the conformational changes induced by rottlerin on the fibril in terms of surface hydrophobicity and secondary structure through 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence and FTIR study respectively. We have also attempted to elucidate the type of interaction between HEWL and rottlerin at pH-12.2 employing techniques like quenching study and FTIR. General significanceRottlerin seems to have potential application as anti-amyloid compound.

Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT(red)) while oxidized dithiothreitol (DTT(ox)) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT(red) was added within 8 days of incubation. Our results showed that the initial addition of DTT(red) interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT(red)-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.

Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme

This study examined the effects of glutathione on the fibrillation of hen egg-white lysozyme. We found that the fibrillation of lysozyme was considerably reduced by GSH while no anti-aggregating activity was detected with only GSSG. SDS-PAGE results also revealed that the addition of GSH led to an early occurrence of prominent lysozyme hydrolysis. Moreover, GSH was effective in inhibiting lysozyme fibrillation when GSH was added within 6 days of incubation. We conclude that the attenuation of lysozyme fibrillation is strongly dependent upon the redox environment. Our data may contribute to decipher the molecular mechanism of amyloid fibrillation.

Investigating the influences of redox buffer compositions on the amyloid fibrillogenesis of hen egg-white lysozyme

More than twenty different human proteins have been found to fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The present study is aimed at exploring the effect of the ratio of cysteine and cystine in the buffer on the fibrillation of hen egg-white lysozyme. Our results revealed that the inhibition of lysozyme amyloid formation by cysteine in the redox buffer followed a concentration-dependent fashion. Cystine, the oxidized form of cysteine, nevertheless, did not influence the final level of fibrillation although it lengthened the lag period of fibril formation. Moreover, the effect of the ratio of cysteine to cystine in the buffer on the fibrillogenesis of hen lysozyme was found to be greatly associated with the formation of mixed disulfide derivatives. Finally, a possible reaction mechanism was proposed to explain our experimental results. Our study shows that the concentration of mixed disulfide derivative was inversely correlated with the level of lysozyme fibrillogenesis. The results from this work may aid in comprehending the molecular mechanism(s) of amyloid fibrillogenesis for disulfide bonded proteins and the development of effective therapeutics for amyloidogenic diseases.

Hen lysozyme amyloid fibrils induce aggregation of erythrocytes and lipid vesicles

We have studied the interaction of hen egg white lysozyme (HEWL) amyloid fibrils with human erythrocytes and lipid vesicles. The fibrils caused extensive aggregation of human erythrocytes and lipid vesicles without any significant lysis. The membrane activity of HEWL fibrils suggests that the interaction of lysozyme fibrils with cellular membranes could be a contributing factor under disease conditions.

Amyloid fibrillation of hen egg-white lysozyme is inhibited by TCEP

This work examines the inhibitory effect of TCEP on the in vitro fibrillation of hen lysozyme at pH 2. We demonstrate that the inhibition of hen lysozyme fibrillation by TCEP follows a dose-dependent manner. Our data show that the addition of TCEP prevents α-to-β transition and promoted unfolding of lysozyme. Moreover, our findings suggested that the TCEP-induced attenuated fibrillation is associated with disulfide disruption and structural unfolding of HEWL.

Effects of Flow on Hen Egg White Lysozyme (HEWL) Fibril Formation: Length Distribution, Flexibility, and Kinetics

The effect of steady shear and turbulent flow on the formation of amyloid fibrils from hen egg white lysozyme (HEWL) was studied. The conversion and size distribution of fibrils obtained by heating HEWL solutions at pH 2 were determined. The formation of fibrils was quantified using flow-induced birefringence. The size distribution was fitted using decay of birefringence measurements and transmission electron microscopy (TEM). The morphology of HEWL fibrils and the kinetics of their formation varied considerably depending on the flow applied. With increasing shear or stirring rate, more rod-like and shorter fibrils were obtained, and the conversion into fibrils was increased. The size distribution and final fibril concentration were significantly different from those obtained in the same heat treatment at rest. The width of the length distribution of fibrils was influenced by the homogeneity of the flow.

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross-beta structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep-UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far-UV CD because it is (i) more sensitive to beta -sheet than to alpha -helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light-scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000-cm(-1) band at various stages of fibrillation.

Inhibition of amyloid fibrillation of lysozyme by indole derivatives − possible mechanism of action

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. There is a great deal of interest in developing small molecule inhibitors of the amyloidogenic processes. In the present article, the inhibitory effects of some indole derivatives on amyloid fibrillation of hen egg white lysozyme (HEWL) are reported. Acidic pH and high temperatures were used to drive HEWL towards amyloid formation. A variety of techniques, ranging from thioflavin T fluorescence and Congo red absorbance assays to far-UV CD and transmission electron microscopy, were employed to characterize the HEWL fibrillation process. Among the indole derivatives tested, indole 3-acetic acid, indole 3-carbinol and tryptophol had the most inhibitory effects on amyloid formation, indole and indole 3-propionic acid gave some inhibition, and indole aldehyde and tryptophan showed no significant inhibition. Although indoles did not protect the HEWL native state from conformational changes, they were effective in diminishing HEWL amyloid fibril formation, delaying both the nucleation and elongation phases. Disaggregation of previously formed HEWL amyloid fibrils was also enhanced by indole 3-acetic acid. Various medium conditions, such as the presence of different anions and alcoholic cosolvents, were explored to gain an insight into possible mechanisms. These observations, taken together, suggest that the indole ring is likely to play the main role in inhibition and that the side chain hydroxyl group may contribute positively, in contrast to the side chain carbonyl and intervening methylene groups.

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition.

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution-alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.

X-ray Scattering Study of the Effect of Hydration on the Cross-β Structure of Amyloid Fibrils

We have investigated the effect of sample hydration on the wide-angle X-ray scattering patterns of amyloid fibrils from two different sources, hen egg white lysozyme (HEWL) and an 11-residue peptide taken from the sequence of transthyretin (TTR105-115). Both samples show an inter-strand reflection at 4.7 A and an inter-sheet reflection which occurs at 8.8 and approximately 10 A for TTR105-115 and HEWL fibrils, respectively. The positions, widths, and relative intensities of these reflections are conserved in patterns obtained from dried stalks and hydrated samples over a range of fibril concentrations. In 2D scattering patterns obtained from flow-aligned hydrated samples, the inter-strand and inter-sheet reflections showed, respectively, axial and equatorial alignment relative to the fibril axis, characteristic of the cross-beta structure. Our results show that the cross-beta structure of the fibrils is not a product of the dehydrating conditions typically employed to produce aligned samples, but is conserved in individual fibrils in hydrated samples under dilute conditions comparable to those associated with other biophysical and spectroscopic techniques. This suggests a structure consisting of a stack of two or more sheets whose interfaces are inaccessible to bulk water.

P.5.016 Prevalence of tardive movement subsyndromes in southern region of Israel

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders, although the relative degree of this functional impairment and the mechanism of selective vulnerability in different regions of the brain related to various neurological diseases are not completely understood. In a recent study, we reported on brain mitochondrial membrane permeabilization upon interaction with hen egg white lysozyme (HEWL) protofibrils, and came to the conclusion that mitochondrial heterogeneity could offer an explanation for some of our observations. Accordingly, the first part of the present investigation was devoted on studies involving interaction of HEWL fibrillation products with mitochondria isolated from various areas of the brain, known to be affected in a number of well-characterized neurodegenerative conditions. This was followed by looking at heart and liver mitochondria. Membrane permeabilization was investigated by monitoring release of mitochondrial enzymes. Mitochondria isolated from cortex and hippocampus showed greater sensitivities than those prepared from substantia nigra. Results clearly demonstrate heterogeneity in brain mitochondria together with a higher resistance to permeabilization in these organelles in comparison with those isolated from liver and heart. Calcium and spermine were found to be more effective in preventing permeabilization in brain mitochondria, as compared to liver and heart. The structure–function aspects and physiological significance of the observations in relation to differences in composition, biophysical nature and morphological properties of mitochondria are discussed. It is argued that studies on heterogeneity in cellular membranes, the primary targets of toxic protofibrils, may provide important insights into mechanism of toxicity, with clinical and pathological manifestations.