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.     

Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin–lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 °C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

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.     

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.     

Protein Fibrillation Lag Times During Kinetic Inhibition

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer’s and Parkinson’s diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.     

Protein Fibrillation Lag Times During Kinetic Inhibition

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer’s and Parkinson’s diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.     

High-pressure refolding of disulfide-cross-linked lysozyme aggregates: thermodynamics and optimization

Previous exploratory work revealed that high pressure (200 MPa), in combination with oxido-shuffling agents such as glutathione, effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules, at concentrations up to 2 mg/mL (1). To understand further and optimize this process, in the current study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH). Yields decreased dramatically at more oxidizing conditions ([GSSG] > [GSH]). Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions. At GSSG:GSH ratios of 4:1, 1:1, and 1:16, lysozyme dissolved and refolded with time constants of 62, 20, and 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.     

Exogenous GSH protection during hypoxia-reoxygenation of the isolated rat heart: impact of hypoxia duration

The objective of this study was to determine the interaction between duration of myocardial hypoxia and presence of exogenous glutathione (GSH) on functional recovery upon subsequent reoxygenation. Isolated perfused rat hearts were subjected to 20, 30, 40, or 50 min hypoxia (HYP), which resulted in a progressive decline in the amount of contractile recovery (% of normoxic rate-pressure product (RPP) and developed pressure) during 30 min reoxygenation. Supplementation with 5 mM GSH throughout normoxia, hypoxia, and reoxygenation significantly improved contractile recovery during reoxygenation after 20 and 30 min hypoxia (p < 0.05), but had no effect after longer durations of hypoxia when contractile recovery was typically below 40% of RPP and significant areas of no-reflow were observed. ECG analysis revealed that GSH shifted the bell-shaped curve for reperfusion ventricular fibrillation to the right resulting in attenuated fibrillation after 20 and 30 min hypoxia then increased incidences after 40 min when Control hearts were slow to resume electrical activity. ECG conduction velocity was well preserved in all hearts after 20 and 30 min hypoxia, but GSH administration significantly attenuated the decline that occurred with longer durations. GSH supplementation did not attenuate the 35% decline in intracellular thiols during 30 min of hypoxia. When 5 mM GSH was added only during 40 min of hypoxia, RPP recovery after reoxygenation was improved compared to unsupplemented Controls (73% vs. 55% of pre-hypoxia value, p < 0.05). Administration of GSH only during reoxygenation following 40 min of hypoxia did not alter RPP recovery compared to Control hearts. We conclude that cardioprotection by exogenous GSH is dependent on the duration of hypoxia and the functional parameter being evaluated. It is not due to an enhancement of intracellular GSH suggesting that exogenous GSH acts extracellularly to protect sarcolemmal proteins against thiol oxidation during the phase of hypoxia when oxidative stress is a major contributor to cardiac dysfunction. Furthermore, if enough damage accrues during oxygen deprivation, supplementing with GSH during reoxygenation will not impact recovery.     

Lysozyme fibrillation: deep UV Raman spectroscopic characterization of protein structural transformation

Deep ultraviolet resonance Raman spectroscopy was demonstrated to be a powerful tool for structural characterization of protein at all stages of fibril formation. The evolution of the protein secondary structure as well as the local environment of phenylalanine, a natural deep ultraviolet Raman marker, was documented for the fibrillation of lysozyme. Concentration-independent irreversible helix melting was quantitatively characterized as the first step of the fibrillation. The native lysozyme composed initially of 32% helix transforms monoexponentially to an unfolded intermediate with 6% helix with a characteristic time of 29 h. The local environment of phenylalanine residues changes concomitantly with the secondary structure transformation. The phenylalanine residues in lysozyme fibrils are accessible to solvent in contrast to those in the native protein.     

Exogenous GSH protection during hypoxia-reoxygenation of the isolated rat heart: Impact of hypoxia duration

The objective of this study was to determine the interaction between duration of myocardial hypoxia and presence of exogenous glutathione (GSH) on functional recovery upon subsequent reoxygenation. Isolated perfused rat hearts were subjected to 20, 30, 40, or 50 min hypoxia (HYP), which resulted in a progressive decline in the amount of contractile recovery (% of normoxic rate-pressure product (RPP) and developed pressure) during 30 min reoxygenation. Supplementation with 5 mM GSH throughout normoxia, hypoxia, and reoxygenation significantly improved contractile recovery during reoxygenation after 20 and 30 min hypoxia (p < 0.05), but had no effect after longer durations of hypoxia when contractile recovery was typically below 40% of RPP and significant areas of no-reflow were observed. ECG analysis revealed that GSH shifted the bell-shaped curve for reperfusion ventricular fibrillation to the right resulting in attenuated fibrillation after 20 and 30 min hypoxia then increased incidences after 40 min when Control hearts were slow to resume electrical activity. ECG conduction velocity was well preserved in all hearts after 20 and 30 min hypoxia, but GSH administration significantly attenuated the decline that occurred with longer durations. GSH supplementation did not attenuate the 35% decline in intracellular thiols during 30 min of hypoxia. When 5 mM GSH was added only during 40 min of hypoxia, RPP recovery after reoxygenation was improved compared to unsupplemented Controls (73% vs. 55% of pre-hypoxia value, p < 0.05). Administration of GSH only during reoxygenation following 40 min of hypoxia did not alter RPP recovery compared to Control hearts. We conclude that cardioprotection by exogenous GSH is dependent on the duration of hypoxia and the functional parameter being evaluated. It is not due to an enhancement of intracellular GSH suggesting that exogenous GSH acts extracellularly to protect sarcolemmal proteins against thiol oxidation during the phase of hypoxia when oxidative stress is a major contributor to cardiac dysfunction. Furthermore, if enough damage accrues during oxygen deprivation, supplementing with GSH during reoxygenation will not impact recovery.     

Fibrillation of hen egg white lysozyme triggers reduction of copper(II)

Copper is known to exert diverse effects on the self-association of proteins and has been found in amyloid deposits that are involved in neurodegenerative disease processes. The effects of the metal ion on the protein during fibrillation were investigated by fluorescence, circular dichroism spectroscopy and fluorescence microscopy. We report for the first time, the complete reduction of Cu(II)→Cu(I) in vitro during fibrillation of hen egg white lysozyme at pH 7. This was confirmed by the lack of any signal for Cu(II) in electron paramagnetic resonance spectroscopy and quantification of Cu(I) was achieved by a bathocuproine disulfonate based assay.     

Anti-fibrillation propensity of a flavonoid baicalein against the fibrils of hen egg white lysozyme: potential therapeutics for lysozyme amyloidosis

More than 20 human diseases involve the fibrillation of a specific protein/peptide which forms pathological deposits at various sites. Hereditary lysozyme amyloidosis is a systemic disorder which mostly affects liver, spleen and kidney. This conformational disorder is featured by lysozyme fibril formation. In vivo lysozyme fibrillation was simulated under in vitro conditions using a strong denaturant GdHCl at 3 M concentration. Sharp decline in the ANS fluorescence intensity compared to the partially unfolded states, almost 20-fold increase in ThT fluorescence intensity, increase in absorbance at 450 nm suggesting turbidity, negative ellipticity peak in the far-UVCD at 217 nm, red shift of 50 nm compared to the native state in Congo red assay and appearance of a network of long rope-like fibrils in transmission electron microscope (TEM) analysis suggested HEWL fibrillation. Anti-fibrillation potency of baicalein against the preformed fibrils of HEWL was investigated following ThT assay in which there was a dose-dependent decrease in ThT fluorescence intensity compared to the fibrillar state of HEWL with the maximum effect observed at 150-μM baicalein concentration, loss of negative ellipticity peak in the far-UVCD region, dip in the Rayleigh scattering intensity and absorbance at 350 and 450 nm, respectively, together with a reduction in the density of fibrillar structure in TEM imaging. Thus, it could be suggested that baicalein could prove to be a positive therapeutics for hereditary human lysozyme amyloidosis.     

General self-assembly mechanism converting hydrolyzed globular proteins into giant multistranded amyloid ribbons

We report a rationale for the formation of amyloid fibrils from globular proteins, and we infer about its possible generality by showing the formation of giant multistranded twisted and helical ribbons from both lysozyme and β-lactoglobulin. We follow the kinetics of the fibrillation under the same conditions of temperature (90 °C) and incubation time (0-30 h) for both proteins, and we assess the structural changes during fibrillation by single-molecule atomic force microscopy (AFM), circular dichroism (CD), and SDS-PAGE. With incubation time, the width of a multistranded fibril increases up to an unprecedented size, with a lateral assembly of as many as 17 protofilaments (173 nm width). In both cases, a progressive unfolding and hydrolysis of the proteins into very short peptide sequences occurs. The molecular weights of peptide fragments, the secondary structure evolution, and the morphology of the final fibrils present striking similarities between lysozyme and β-lactoglobulin. Because of additional analogies to synthetic peptide fibrils, these findings support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role.     

High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time

Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0–5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ∼3.0 m, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.     

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.     

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

Background

More 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).

Methods

ThT 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 conclusion

The 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 significance

We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.     

The role of protonation in protein fibrillation

Many proteins fibrillate at low pH despite a high population of charged side chains. Therefore exchange of protons between the fibrillating peptide and its surroundings may play an important role in fibrillation. Here, we use isothermal titration calorimetry to measure exchange of protons between buffer and the peptide hormone glucagon during fibrillation. Glucagon absorbs or releases protons to an extent which allows it to attain a net charge of zero in the fibrillar state, both at acidic and basic pH. Similar results are obtained for lysozyme. This suggests that side chain pK_a values change dramatically in the fibrillar state.     

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 μM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.