Cumulative asparagine to aspartate deamidation fails to perturb γD-crystallin structure and stability.

Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1H-15N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation.

Participation of Lens Proteins and miRs in Traumatic and Inheritance Cataract: A Review on Diagnostic and Therapeutic Approaches for Cataract Management.

Traumatic and inherited cataract spiking blindness is caused by accumulated deposition of mutant eye lens protein or lens microarchitecture alteration. A traumatic cataract is a clouding of the eye's natural lens that occurs as a result of physical trauma to the eye. This trauma can be caused by various incidents such as blunt force injury, penetration by a foreign object, or a significant impact on the eye area. Inheritance cataracts or hereditary cataracts are cataracts that are genetically inherited from one or both parents. Complications following cataract surgery encompass various adverse outcomes such as inflammation, infection, bleeding, swelling, drooping eyelid, glaucoma, secondary cataracts, and complete loss of vision. The main purpose of the review is to highlight common pathophysiology associated with traumatic and inherited cataracts. Also, the review discusses diagnosis and treatment strategies for such cataract types by targeting their key pathological hallmarks. γD-crystallin plays a crucial role in maintaining the optical properties of the lens during the life span of an individual. Carbamazepine, Resveratrol, and Myricetin (CRM) are effectively bound at the γD-crystallin binding site and thereby could minimize misfolding and aggregation of γD-crystallin. miR-202, miR-193b, miR-135a, miR365, and miR-376a had the highest levels of abundance in the aqueous humor of individuals diagnosed with cataracts. The validation of these miRs will provide more insights into their functional roles and may be used for diagnostic purposes. The effective CRM combination as a multidrug formulation may postpone both traumatic and inherited cataracts and protect the eye from blindness.

Cataract-causing mutations S78F and S78P of γD-crystallin decrease protein conformational stability and drive aggregation.

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.

The mechanism of self-association of human γ-D crystallin from molecular dynamics simulations.

The aggregation of human γ-D crystallin is associated with the age-onset cataract formation. Here, we extensively investigated the self-association mechanism of human γ-D crystallin through molecular dynamics computer simulations. By mutating the protein surface we found that electrostatic interactions between charged amino acids play a crucial role in its self-association. We have confirmed the two-fold role of arginine molecules. If they are located as residues on the protein surface they can initiate protein contacts and contribute to their stickiness with noteworthy hydrophobic interactions through stacking of their methylene groups. But if they are added as free arginine in the protein solution they can also stabilize it, by associating with the protein surface and also with themselves to form effective inter-protein spacers that obstruct protein aggregation.

Insights to Human γD-Crystallin Unfolding by NMR Spectroscopy and Molecular Dynamics Simulations.

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis.

The contribution of individual residues of an aggregative hexapeptide derived from the human γD-crystallin to its amyloidogenicity.

Human γD-crystallin protein is abundant in the lens and is essential for preserving lens transparency. With age the protein may lose its native structure resulting in the formation of cataract. We recently reported an aggregative peptide, 41Gly-Cys-Trp-Met-Leu-Tyr46 from the human γD-crystallin, termed GDC6, exhibiting amyloidogenic properties in vitro. Here, we aimed to determine the contribution of each residue of the GDC6 to its amyloidogenicity. Molecular dynamic (MD) simulations revealed that the residues Trp, Leu, and Tyr played an important role in the amyloidogenicity of GDC6 by facilitating inter-peptide main-chain hydrogen bonds, and π-π interactions. MD predictions were further validated using single-, double- and triple-alanine-substituted GDC6 peptides in which their amyloidogenic propensity was individually evaluated using complementary biophysical techniques including Thioflavin T assay, turbidity assay, CD spectroscopy, and TEM imaging. Results revealed that the substitution of Trp, Leu, and Tyr together by Ala completely abolished aggregation of GDC6 in vitro, highlighting their importance in the amyloidogenicity of GDC6.

Insights into the binding of morin to human γD-crystallin.

Crystallin aggregation in the eye lens is one of the leading causes of cataract formation. The increase in the human γD-crystallin (HγDC) aggregation propensity has been associated with the oligomerization of its partially folded and fully unfolded structure. A recent study demonstrated that the binding of flavonoid morin (MOR) to HγDC inhibits the fibrillation of this protein. In this work, we carry out an exhaustive search for the possible binding site of MOR on HγDC by combining an ensemble docking approach with the Wrap 'N' Shake protocol. In agreement with previous results, we found a potential MOR-binding site in the cleft formed between the N-terminal and C-terminal domains of HγDC. MOR preference for the cleft residues was observed even with the interface-opened intermediate conformers of HγDC. In addition, metadynamics simulations were carried out to corroborate the stabilizing activity of MOR on HγDC structure and to identify the structural regions implicated during the unfolding inhibition. Overall, this study provides relevant insights into the identification of new HγDC aggregation inhibitors.

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.

Inhibition of amyloid fibrillation of γD-crystallin model peptide by the cochineal Carmine.

γD-crystallin is among the most abundant γ-crystallins in the human eye lens which are essential for preserving its transparency. Aging, and environmental changes, cause crystallins to lose their native soluble structure and aggregate, resulting in the formation of cataract. Current treatment of cataract is surgical removal which is costly. Pharmaceutical therapeutics of cataract is an unmet need. We report a screen for small molecules capable of inhibiting aggregation of human γD-crystallin. Using a highly amyloidogenic hexapeptide model 41GCWMLY46 derived from the full-length protein, we screened a library of 68 anthraquinone molecules using ThT fluorescence assay. A leading hit, the cochineal Carmine, effectively reduced aggregation of the model GDC6 peptide in dose dependent manner. Similar effect was observed toward aggregation of the full-length γD-crystallin. Transmission electron microscopy, intrinsic Tryptophan fluorescence and ANS fluorescence assays corroborated these results. Insights obtained from molecular docking suggested that Carmine interaction with monomeric GDC6 involved hydrogen bonding with Ace group, Cys, Met residues and hydrophobic contact with Trp residue. Carmine was non-toxic toward retinal cells in culture. It also reduced ex vivo the turbidity of human extracted cataract material. Collectively, our results indicate that Carmine could be used for developing new therapeutics to treat cataract.

Inhibition of fibrillation of human γd-crystallin by a flavonoid morin.

To inhibit the formation of amyloid fibrils by human γd-crystallin (HGD), a series of four flavonoids (quercertin, rutin, morin and hesperetin) was tested. Only morin had demonstrated significant inhibition of HGD fibrillation. Results from fluorimetric assay techniques (using thioflavin T and ANS), FTIR, circular dichroism and microscopic imaging (fluorescence microscopy and transmission electron microscopy) confirmed HGD fibrillation inhibition by morin. HGD-morin complex formation at ground state resulted tryptophan fluorescence quenching through static mechanism, which was also confirmed by determining the excited-state life time of HGD tryptophan residues. Förster resonance energy transfer occurs from HGD to morin. Synchronous, three-dimensional fluorescence, FTIR and circular dichroism results suggest that major changes in HGD conformation did not occur on binding with morin. The interactions between HGD and morin involve hydrogen bonding and/or van der Waals forces. Docking predictions also support experimental results.Communicated by Ramaswamy H. Sarma.

Elucidation of molecular interactions between human γD-crystallin and quercetin, an inhibitor against tryptophan oxidation.

Different post-translational changes in eye lens crystallin proteins contribute towards the development of cataract. We have studied in vitro oxidative modification of tryptophan (Trp) residues of human γD-crystallin (HGD) towards formation of N-formylkynurenine (NFK) associated with cataractogenesis. This oxidation was found to be inhibited by quercetin at relatively low concentration. Interactions between quercetin and HGD were further studied using fluorescence techniques. Binding and quenching constants were determined as ∼104 M-1. Static quenching of fluorescence due to HGD-quercetin complex formation at ground state was confirmed by finding excited state life time of Trp residues. Energy transfer occurred between the protein and quercetin. Hydrogen bonding and/or van der Waals interactions were involved between HGD and quercetin. Synchronous and three-dimensional fluorescence along with far-UV CD studies suggested no major conformational alterations occurred in HGD due to quercetin binding. Experimental observations were supported by the docking results.Communicated by Ramaswamy H. Sarma.

Assessing the Structures and Interactions of γD-Crystallin Deamidation Variants.

Cataracts involve the deposition of the crystallin proteins in the vertebrate eye lens, causing opacification and blindness. They are associated with either genetic mutation or protein damage that accumulates over the lifetime of the organism. Deamidation of Asn residues in several different crystallins has been observed and is frequently invoked as a cause of cataract. Here, we investigated the properties of Asp variants, deamidation products of γD-crystallin, by solution NMR, X-ray crystallography, and other biophysical techniques. No substantive structural or stability changes were noted for all seven Asn to Asp γD-crystallins. Importantly, no changes in diffusion interaction behavior could be detected. Our combined experimental results demonstrate that introduction of single Asp residues on the surface of γD-crystallin by deamidation is unlikely to be the driver of cataract formation in the eye lens.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

An amyloidogenic hexapeptide from the cataract-associated γD-crystallin is a model for the full-length protein and is inhibited by naphthoquinone-tryptophan hybrids.

The eye lens is rich in proteins called crystallins, whose native conformation is crucial for preserving its transparency. With aging, crystallins may be exposed to environmental changes, which could lead to their aggregation and eventually to cataract development. Human γD-crystallin, among the most abundantly expressed γ-crystallins in the lens, was shown to form amyloid aggregates under denaturing conditions in vitro. However, the exact mechanism of aggregation remains to be clearly defined. Here, using prediction algorithms and biophysical methods, we identified a hexapeptide 41GCWMLY46 as a most aggregative fragment in human γD-crystallin. Two aromatic naphthoquinone-tryptophan hybrid molecules (NQTrp and Cl-NQTrp), inhibited the in vitro aggregation of this hexapeptide as well as full-length γD-crystallin in a dose-dependent manner, plausibly facilitated by hydrogen bonding and aromatic contacts with the hydrophobic residues. The two compounds had no toxic effect toward retinal cell culture and reduced the cytotoxicity induced by aggregates of the hexapeptide. In addition, NQTrp and Cl-NQTrp were able to disassemble pre-formed aggregates of the hexapeptide and the full-length γD-crystallin. Our results indicate that the amyloidogenic hexapeptide is a useful model for screening inhibitors of γD-crystallin and that the NQTrp hybrid scaffolds may serve as leads for developing new drugs for treating cataract.

Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level.

The capability of citrate-stabilized gold nanoparticles (AuNps) has been explored for the inhibition of amyloid fibrillation of human γD-crystallin (HGD), a major protein of eye lens. Citrate-capped AuNps were synthesized, characterized and used further for amyloid inhibition. The results from intrinsic and extrinsic (in the presence of Thioflavin T and ANS) fluorescence based assays and CD spectroscopy clearly suggest that AuNps at nanomolar concentrations can act as an effective inhibitor against fibrillation of HGD. Fluorescence microscopic and transmission electron microscopic images also supported this observation. Considering the inhibitory role of AuNps against HGD fibrillation, interactions between HGD and AuNps were studied to decipher the mechanism of amyloid inhibition. The binding and quenching constants were calculated as ~109 M-1 using the data of tryptophan fluorescence quenching of HGD by AuNps. Ground state complexation between the protein and nanoparticles was predicted. AuNps were not found to cause any major conformational changes in the native protein. Entropy-driven complexation process between the protein and nanoparticles indicates the interactions of AuNps with hydrophobic residues of HGD. Therefore, in the presence of AuNps, the exposure of the hydrophobic patches of HGD during its partial unfolding became restricted, which results inhibition in HGD fibrillation.

Protective role of hesperetin against posttranslational oxidation of tryptophan residue of human γD-crystallin: A molecular level study.

Crystallin proteins undergo various posttranslational modifications with aging of eye lens. Oxidation of tryptophan (Trp) residues of a major γ-crystallin namely human γD-crystallin (HGD) was found to be inhibited by a naturally occurring flavonoid hesperetin at relatively low concentration mostly due to its antioxidant activity. Further the molecular interactions between HGD and hesperetin were elucidated on the basis of the quenching of Trp fluorescence of the protein by the flavonoid. Ground state complexation between HGD and hesperetin caused static quenching of the Trp fluorescence of HGD. Binding and quenching constants were in the order of (103- 104 M-1). Energy transfer from protein to hesperetin was suggested by FRET calculations. Thermodynamic parameters reveal significant hydrophobic association between the protein and hesperetin. Synchronous fluorescence and CD spectroscopic results had ruled out conformational changes in the protein due to binding of hesperetin. Docking studies suggested the proximity of hesperetin with Trp 42, which largely corroborates our experimental findings.

Crystallin Fusion Proteins Improve the Thermal Properties of Hair.

Styling hair with straightening irons is a popular daily hair routine that significantly damage the hair keratin fiber due to the high temperature applied. In this study, we investigate the effect of two fusion proteins based on the human eye γD-crystallin conjugated with a keratin-based peptide (KP-Cryst Wt and KP-Cryst Mut) on hair exposed to thermal damage. The mutant form was designed to improve protein stability and promote interaction with the hair. Through the study, it was demonstrated the protection of Asian and Caucasian virgin hair's structure by the pretreatments with the KP-Cryst fusion proteins. After hair thermal exposure, a higher water content was quantified by TGA on the hair fibers pretreated with the fusion proteins (about 38% for the KP-Cryst Wt and 44% for the KP-Cryst Mut). Also, negligible alterations in hair fibers' stiffness were observed after iron application, demonstrating the proteins capacity to effectively prevent the conversion of keratin α-helix structure into ß-sheets. The results proved the capacity of the fusion proteins to bind to hair and protect it against high temperatures', supporting the development of new formulations based on the KP-Cryst proteins.

Inhibition of copper-induced aggregation of human γD-crystallin by rutin and studies on its role in molecular level for enhancing the chaperone activity of human αA-crystallin by using multi-spectroscopic techniques.

Oxidative aggregation of γ-crystallins induced by copper in aged lens increases the lens opacity and causes cataract formation. Therefore, chelation of free Cu2+ by small molecules can inhibit metal-mediated aggregation of γ-crystallin. In this work, the inhibition potency of several naturally occurring flavonoid compounds was studied against aggregation of human γD-crystallin (HGD) mediated by copper ions. Among them, rutin demonstrated ~20% inhibition of HGD aggregation induced by Cu2+ through its metal chelation ability. Not only that, the chaperone activity of lens chaperone, human αA-crystallin (HAA) was found to be enhanced in the presence of rutin. Subsequently, the molecular interactions between HAA and rutin were investigated using fluorescence and CD spectroscopy to understand the molecular basis of the chaperone activity enhancement by rutin. Quenching of HAA fluorescence by rutin with a quenching constant in the order of ~105 M-1 depicts a complexation between them. Entropy driven process of complexation between HAA and rutin suggests significant involvement of hydrophobic interactions. Fluorescence resonance energy transfer between protein and ligand can occur at a distance of 2.73 nm. Synchronous fluorescence and circular dichroism spectroscopy revealed that protein-ligand interaction does not cause any notable conformational changes in HAA. Experimental observations have been well substantiated by docking.

A molecular dynamics approach to explore the structural characterization of cataract causing mutation R58H on human γD crystallin.

The crystallins are a family of monomeric proteins present in the mammalian lens and mutations in these proteins cause various forms of cataracts. The aim of our current study is to emphasize the structural characterization of aggregation propensity of mutation R58H on γD crystallin using molecular dynamics (MD) approach. MD result revealed that difference in the sequence level display a wide variation in the backbone atomic position, and thus exhibits rigid conformational dynamics. Changes in the flexibility of residues favoured to increase the number of intra-molecular hydrogen bonds in mutant R58H. Moreover, notable changes in the hydrogen bonding interaction resulted to cause the misfolding of mutant R58H by introducing α-helix. Principal component analysis (PCA) result suggested that mutant R58H showed unusual conformational dynamics along the two principal components when compared to the wild-type (WT)-γD crystallin. In a nutshell, the increased surface hydrophobicity could be the cause of self-aggregation of mutant R58H leading to aculeiform cataract.

Inhibition of amyloid fibrillation and destabilization of fibrils of human γD-crystallin by direct red 80 and orange G.

Inhibition of amyloid fibril formation by a lens protein namely human γD-crystallin (HGD) under stressful conditions was targeted by using some small molecules like direct red 80 (DR), orange G (OG) and rhodamine B (RH). The protein itself was found to form matured fibrils after 48h of incubation at pH 3.0 at 37°C. Various fluorescence based assays (thioflavin T assay, ANS binding assay, intrinsic Trp fluorescence determination), circular dichroism and microscopic imaging techniques were used in the inhibition studies. Above studies unequivocally proved that DR had acted as the most potent inhibitor among these molecules and it was little better efficient than OG. RH had shown a moderate inhibition of HGD fibrillation. Microscopic images from fluorescence microscopy and transmission electron microscopy also substantiated our spectroscopic observations. These small molecules were not only capable to restrict the fibrillation, but they were also able to disassemble the mature and premature fibrils of HGD. Hydrophobic and aromatic interactions between the inhibitor molecules and partially unfolded HGD are likely to be responsible for exhibiting inhibition of protein fibrillation.