Protection of human γD-crystallin protein from ultraviolet C-induced aggregation by ortho-vanillin.

Cataract is known as one of the leading causes of vision impairment worldwide. While the detailed mechanism of cataratogenesis remains unclear, cataract is believed to be correlated with the aggregation and/or misfolding of human ocular lens proteins called crystallins. A 173-residue structural protein human γD-crystallin is a major γ-crystallin protein in the human eye lens and associated with the development of juvenile and mature-onset cataracts. This work is aimed at investigating the effect of a small molecule, e.g., ortho-vanillin, on human γD-crystallin aggregation upon exposure to ultraviolet-C irradiation. According to the findings of right-angle light scattering, transmission electron microscopy, and gel electrophoresis, ortho-vanillin was demonstrated to dose-dependently suppress ultraviolet-C-triggered aggregation of human γD-crystallin. Results from the synchronous fluorescence spectroscopy, tryptophan fluorescence quenching, and molecular docking studies revealed the structural change of γD-crystallin induced by the interaction/binding between ortho-vanillin and protein. We believe the outcome from this work may contribute to the development of potential therapeutics for cataract.

Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of ß-lactoglobulin.

In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of ß-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress ß-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better ß-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the ß-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore-nanoparticle complex are involved in the nanoparticle-ß-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.

Examining the effects of dextran-based polymer-coated nanoparticles on amyloid fibrillogenesis of human insulin.

More than thirty human proteins and/or peptides can aggregate to form amyloid deposits that are linked to several amyloid diseases including clinical syndrome injection-localized amyloidosis, which is correlated with the aggregation of the 51-residue polypeptide insulin. While no cure is currently available toward tackling amyloid diseases, prevention or suppression of amyloid fibrillization is considered as the primary therapeutic strategy. Nanomaterials have been demonstrated to possess great potential in the fields of biomedical diagnosis and drug delivery, they are also able to affect the amyloid aggregation of proteins. This work explores the effects of three different magnetic nanoparticles coated with dextran-based polymers on the in vitro amyloid fibrillogenesis of human insulin. Surface modification of nanoparticles with dextran-based polymers was used to improve the biocompatibility of maghemite nanoparticles. We demonstrated that insulin fibrillization may be mitigated by the studied nanoparticles in a concentration-dependent fashion as verified by ThT binding assay and transmission electron microscopy. The extent of inhibitory activity against human insulin fibril formation was found to be associated with the physico-chemical properties of nanoparticles, with the highest inhibitory activity observed for diethylaminoethyl-dextran-coated nanoparticles. Using circular dichroism spectroscopy, ANS fluorescence spectroscopy, and right-angle light scattering, we probed the structural/conformational changes and investigated the aggregating behavior of insulin upon treatment with nanoparticles. This work demonstrates that nanoparticles with an appropriate surface modification can be utilized to suppress or even inhibit amyloid fibril formation of proteins.

Exploring the effects of methylene blue on amyloid fibrillogenesis of lysozyme.

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents.

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Lysozyme amyloid fibrillization in presence of tacrine/acridone-coumarin heterodimers.

Amyloid aggregates of proteins are one of the most abundant and important naturally occurring self-associated assemblies. Formation of poly/peptide amyloid aggregates is also associated with the widely spread diseases, so called amyloidosis, which include Alzheimer's disease, diabetes mellitus and lysozyme amyloidosis. These disorders are still incurable and novel therapeutical approaches are focused on using small molecules for inhibition of amyloid aggregation. We have observed effect of three structurally distinct groups of tacrine/acridone - coumarin heterodimers on hen egg white (HEW) lysozyme fibrillization in vitro. The ability of heterodimers to interfere with lysozyme amyloid aggregation was examined using Thioflavin T fluorescence assay, atomic force microscopy and docking method. The obtained data suggest that inhibitory effect of heterodimers on lysozyme fibrillization depends on their composition. We have shown that tacrine-coumarin heterodimers with alkylenediamine linker are the most effective inhibitors of lysozyme fibrillization. The inhibitory activities were quantified through IC50 values; the most potent heterodimers interfere with lysozyme aggregation in the scale of micromolar concentrations (19.2 µM-105.4 µM). The molecular docking showed that the modes of possible interactions involved in the binding are mainly hydrophobic interactions, hydrogen bonding and van der Waals interactions. Studied heterodimers had none or weak cytotoxic effect on human neuroblastoma cells. The obtained results can be helpful for the design and development of new therapeutics for amyloid-related diseases.

Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

Investigation of the early stages of human γD-crystallin aggregation process.

Cataract, a major cause of visual impairment worldwide, is a common disease of the eye lens related to protein aggregation. Several factors including the exposure of ultraviolet irradiation and possibly acidic condition may induce the unfolding and subsequent aggregation of the crystallin proteins leading to crystalline lens opacification. Human γD-crystallin (HγDC), a 173 residue monomeric protein, abundant in the nucleus of the human eye lens, has been shown to aggregate and form amyloid fibrils under acidic conditions and that this aggregation route is thought to be a potential initiation pathway for the onset of age-related nuclear cataract. However, the underlying mechanism of fibril formation remains elusive. This report is aimed at examining the structural changes and possible amyloid fibril formation pathway of HγDC using molecular dynamics and molecular docking simulations. Our findings demonstrated that incubation of HγDC under the acidic condition redistributes the protein surface charges and affects the protein interaction with its surrounding solvent environment. This brings about a twist motion in the overall tertiary structure that gives rise to newly formed anti-parallel ß-strands in the C-terminal flexible loop regions. The change in protein structural conformation also involves an alteration in specific salt-bridge interactions. Altogether, these findings revealed a plausible mechanism for amyloid fibril formation of HγDC that is important to the early stages of HγDC aggregation involved in cataractogenesis.

Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions.

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye's crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

Combining structure-based pharmacophore and in silico approaches to discover novel selective serotonin reuptake inhibitors.

Inhibition of human serotonin transporter (hSERT) has been reported to be a potent strategy for the treatment for depression. To discover novel selective serotonin reuptake inhibitors (SSRIs), a structure-based pharmacophore model (SBPM) was developed using the docked conformations of six highly active SSRIs. The best SBPM, consisting of four chemical features: two ring aromatics (RAs), one hydrophobic (HY), and one positive ionizable (PI), was further validated using Gunner-Henry (GH) scoring and receiver operating characteristic (ROC) curve methods. This well-validated SBPM was then used as a 3D-query in virtual screening to identify potential hits from National Cancer Institute (NCI) database. These hits were subsequently filtered by absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction and molecular docking, and their binding stabilities were validated by 20-ns MD simulations. Finally, only two compounds (NSC175176 and NSC705841) were identified as potential leads, which exhibited higher binding affinities in comparison with the paroxetine. Our results also suggest that cation-π interaction plays a crucial role in stabilizing the hSERT-inhibitor complex. To our knowledge, the present work is the first structure-based virtual screening study for new SSRI discovery, which should be a useful guide for the rapid identification of novel therapeutic agents from chemical database.

Molecular dynamics simulation of the induced-fit binding process of DNA aptamer and L-argininamide.

Aptamers are rare functional nucleic acids with binding affinity to and specificity for target ligands. Recent experiments have lead to the proposal of an induced-fit binding mechanism for L-argininamide (Arm) and its binding aptamer. However, at the molecular level, this mechanism between the aptamer and its coupled ligand is still poorly understood. The present study used explicit solvent molecular dynamics (MD) simulations to examine the critical bases involved in aptamer-Arm binding and the induced-fit binding process at atomic resolution. The simulation results revealed that the Watson-Crick pair (G10-C16), C9, A12, and C17 bases play important roles in aptamer-Arm binding, and that binding of Arm results in an aptamer conformation optimized through a general induced-fit process. In an aqueous solution, the mechanism has the following characteristic stages: (a) adsorption stage, the Arm anchors to the binding site of aptamer with strong electrostatic interaction; (b) binding stage, the Arm fits into the binding site of aptamer by hydrogen-bond formation; and (c) complex stabilization stage, the hydrogen bonding and electrostatic interactions cooperatively stabilize the complex structure. This study provides dynamics information on the aptamer-ligand induced-fit binding mechanism. The critical bases in aptamer-ligand binding may provide a guideline in aptamer design for molecular recognition engineering.

In silico investigation of the disease-associated retinoschisin C110Y and C219G mutants.

The juvenile X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the secretory protein, retinoschisin (RS1). Majority of the disease is resulted from single point mutations on the RS1 discoidin domain with cysteine mutations being related to some of the more severe cases of XLRS. Previous studies have indicated that two mutations (C110Y and C219G), which involve cysteines that form intramolecular disulfide bonds in the native discoidin domain, resulted in different oligomerization states of the proteins and did not correlate with the degree of protein stability as calculated by the change in folding free energy. Through homology modeling, bioinformatics predictions, molecular dynamics (MD) and docking simulations, we attempt to investigate the effects of these two mutations on the structure of the RS1 discoidin domain in relevance to the discrepancy found between structural stability and aggregation propensity. Based on our findings, this discrepancy can be explained by the ability of C110Y mutant to establish suitable modules for initiating amorphous aggregation and to expand the aggregating mass through predominantly hydrophobic interactions. The low capability of C219G mutant to oligomerize, on the other hand, may be due to its greater structural instability and lesser hydrophobic tendency, two properties that may be unsupportive of aggregation. The results, altogether, indicate that aggregation propensity in the RS1 C110Y mutant is dependent upon the formation of suitable aggregating substrates for propagation of aggregation and not directly related to or determined by overall structural instability. As for the wildtype protein, the binding specificity of the spikes for biological function and the formation of octameric structure are contributed by important loop interactions, as well as evolved structural and sequence-based properties that prevent aggregation.

The possible structural models for polyglutamine aggregation: a molecular dynamics simulations study.

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial ß-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung ß-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high ß-turn and ß-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable ß-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of ß-turn and ß-sheet are sampled and preserved during the very early stage of aggregation.

The discovery of potential acetylcholinesterase inhibitors: a combination of pharmacophore modeling, virtual screening, and molecular docking studies.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD. METHODS: In this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors. RESULTS: The best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R² = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aß toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site. CONCLUSIONS: The information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.

The importance of steric zipper on the aggregation of the MVGGVV peptide derived from the amyloid beta peptide.

Amyloid-like fibrils are found in many fatal diseases, such as Alzheimer's disease, Parkinson's disease, type II diabetes mellitus, and prion diseases. Recently, the structural characterization of the MVGGVV peptide from the C-terminal hydrophobic segment of the amyloid-B (AB) peptide has revealed a general feature of amyloid-like fibrils, termed as "steric zipper", which is constituted by a tight side-chain complementation of the opposing B-sheet layers. In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the importance of steric zipper on the aggregation of the MVGGVV peptide. Our results show that the structural stability of the MVGGVV oligomers increases with increasing the number of B-strands. We further proposed that the octameric structure (the SH2-ST4 model in this study) is the possible nucleus seed for MVGGVV protofibril formation. Our results also demonstrated that hydrophobic interaction is the principle driving force to stabilize the adjacent B-strands while the steric zipper involved M1, V2, V5 and V6 is responsible for holding the neighboring B-sheet layers together. Finally, a twisted model of the MVGGVV assembly (SH2-ST50), based on the averaged twisted angle of approximately 11.5 degrees between the adjacent B-strands of the SH2-ST4 model, was proposed. Our results gain insights into the aggregation of the MVGGVV peptide in atomic details and may provide a hint for designing new inhibitors able to prevent the fibril formation of the AB peptide.

Diseases of protein aggregation and the hunt for potential pharmacological agents.

Protein aggregation is a ubiquitous phenomenon significant to all aspects of science. Notably, the formation of protein aggregates is frequently encountered in biochemical research and biopharmaceutical industry. Formation of protein aggregates is generally regarded to be associated with partially folded intermediate species that are susceptible to self-association due to the exposure of hydrophobic core. Evidence supports the concept that the formation of aggregates in vitro is a generic property of proteins. In human etiology, more than 20 different devastating human diseases have been reported to be associated with protein aggregation. Although protein aggregation diseases have been the center of intense research, much remains to be learned regarding the underlying molecular mechanisms. In this review, the general background information on protein aggregation is first provided. Next, we summarize the properties, characteristics and causes of protein aggregates. Finally, from the perspectives of epidemiology, pathogenesis, existing mechanisms, relevant hypotheses, and current as well as potential therapeutic approaches, two examples of protein aggregation diseases, Alzheimer's disease and cataract, are briefly discussed. Importantly, while a variety of molecules have been suggested, the effective therapeutic drugs for curing the diseases involving protein aggregation have yet to be identified. We believe that a better understanding of the mechanisms of protein aggregation process and an extensive investigation into the drug penetration, efficacy, and side effects will certainly aid in developing the successful pharmacological agents for these diseases.

Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone.

It has been reported that more than 20 different human proteins can 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 current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p-benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p-benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p-benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.