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Congenital cataracts, a prevalent cause of blindness in children, are associated with protein aggregation. γD-crystallin, essential for sustaining lens transparency, exists as a monomer and exhibits excellent structural stability. In our cohort, we identified a nonsense mutation (c.451_452insGACT, p.Y151X) in the CRYGD gene. To explore the effect of truncation mutations on the structure of γD-crystallin, we examined the Y151X and T160RfsX8 mutations, both located in the Greek key motif 4 at the cellular and protein level in this study. Both truncation mutations induced protein misfolding and resulted in the formation of insoluble aggregates when overexpressed in HLE B3 and HEK 293T cells. Moreover, heat, UV irradiation, and oxidative stress increased the proportion of aggregates of mutants in the cells. We next purified γD-crystallin to estimate its structural changes. Truncation mutations led to conformational disruption and a concomitant decrease in protein solubility. Molecular dynamics simulations further demonstrated that partial deletion of the conserved domain within the Greek key motif 4 markedly compromised the overall stability of the protein structure. Finally, co-expression of α-crystallins facilitated the proper folding of truncated mutants and mitigated protein aggregation. In summary, the structural integrity of the Greek key motif 4 in γD-crystallin is crucial for overall structural stability., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiangjun Chen reports financial support was provided by the National Natural Science Foundation of China. Lidan Hu reports financial support was provided by the National Natural Science Foundation of China. Wei Wu reports financial support was provided by the National Natural Science Foundation of China. Xiangjun Chen reports financial support was provided by the Natural Science Foundation of Zhejiang Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

γS-crystallin is particularly rich in the embryonic nuclear region and is crucial to the maintenance of lens transparency and optical properties. Gene mutations in crystallin are the main factors leading to congenital hereditary cataracts, which are a major cause of visual impairment in children. Some mutations located in the 18th amino acid glycine of γS-crystallin were reported to be linking with congenital cataracts. However, the pathogenic mechanism has not been elucidated. Interestingly, we previously identified a novel variant of γS-crystallin (c.53G > A; p. G18D) with progressive cortical and sutural congenital cataracts in one Chinese family. In this study, we purified the γS-crystallin wildtype and mutant proteins to investigate the effects of the G18D mutation on the structural stability of γS-crystallin. The results showed that there were tertiary structural differences between the wild-type γS-crystallin and the G18D variant. The mutation significantly impaired the stability of γS-crystallin under environmental stress and promoted aggregation. Furthermore, molecular dynamics (MD) simulations showed that the mutation altered H-bonding and surface electrostatic potential. Significantly decreased stability along with an increased tendency to aggregate under environmental stress may be the major pathogenic factors for cataracts induced by the G18D mutation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite of increasingly accumulated genetic variations of autosomal dominant congenital cataracts (ADCC), the causative genes of many ADCC patients remains unknown. In this research, we identified a novel F30S mutation in γS-crystallin from a three-generation Chinese family with ADCC. The patients possessing the F30S mutation exhibited nuclear cataract phenotype. The potential molecular mechanism underlying ADCC by the F30S mutation was investigated by comparing the structural features, stability and aggregatory potency of the mutated protein with the wild type protein. Spectroscopic experiments indicated that the F30S mutation did not affect γS-crystallin secondary structure compositions, but modified the microenvironments around aromatic side-chains. Thermal and chemical denaturation studies indicated that the mutation destabilized the protein and increased its aggregatory potency. The mutation altered the two-state unfolding of γS-crystallin to a three-state unfolding with the accumulation of an unfolding intermediate. The almost identical values in the changes of Gibbs free energies for transitions from the native state to intermediate and from the intermediate to unfolded state suggested that the mutation probably disrupted the cooperativity between the two domains during unfolding. Our results expand the genetic variation map of ADCC and provide novel insights into the molecular mechanism underlying ADCC caused by mutations in β/γ-crystallins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Crystallin gene mutations are responsible for about half of the congenital cataract caused by genetic disorders. L45P and Y46D mutations of γC-crystallin have been reported in patients with nuclear congenital cataract. In this study, we explored the thermal stability of wild type (WT), L45P, and Y46D mutants of γC-crystallin at low and high concentrations, as well as the effect of αA-crystallin on the thermal stability of mutants. Spectroscopic experiments were used to monitor the structural changes on temperature-gradient and time-course heating process. Intermediate morphologies were determined through cryo-electron microscopy. The thermal stability of WT and mutants at concentrations ranging up to hundreds of milligrams were assessed via the UNcle multifunctional protein stability analysis system. The results showed that L45P and Y46D mutations impaired the thermal stability of γC-crystallin at low (0.2 mg/mL) and high concentrations (up to 200 mg/mL). Notably, with increase in protein concentration, the thermal stability of L45P and Y46D mutants of γC-crystallin simultaneously decreased. Thermal stability of L45P and Y46D mutants could be rescued by αA-crystallin in a concentration-dependent manner. The dramatic decrease in thermal stability of γC-crystallin caused by L45P and Y46D mutations contributed to congenital cataract in the mature human lens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 41 GCWMLY 46 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

αβγ-crystallins account for ∼90% of ocular proteins in lens with concentrations ≥400 mg/ml, which has to be soluble for the whole life-span and their aggregation results in cataract. So far, four cataract-causing mutants G18V, D26G, S39C and V42 M have been identified for human γS-crystallin. Mysteriously, lens maintains ATP concentrations of 3-7 mM despite being a metabolically-quiescent organ. Here by DSF and NMR, we characterized the binding of ATP to three cataract-causing mutants of human γS-crystallin as well as its effect on the solution conformations and thermal stability. The results together decode several novel findings: 1) ATP shows no detectable binding to WT and mutants, as well as no significant alternation of their conformations even at molar ratio of 1:200.2) Cataract-causing mutants show distinctive patterns of the crowding-induced destabilization. 3) ATP differentially antagonizes their crowding-induced destabilization. Our studies suggest that the crowding-induced destabilization of human γS-crystallin is also critically dependent of the hydration shell which could be differentially altered by four mutations. Most unexpectedly, ATP acts as an effective mediator for the protein hydration shell to antagonize the crowding-induced destabilization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In lens, ∼90% of ocular proteins are αβγ-crystallins with concentrations ≥400 mg/ml, which need to remain soluble for the whole life-span and their aggregation leads to cataract. The G18V mutation of human γS-crystallin causes hereditary childhood-onset cortical cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3-7 mM. Very recently, we found that ATP has no significant binding to γS-crystallin as well as no alternation of its conformation. Nevertheless, ATP antagonizes the crowding-induced destabilization of γS-crystallin even at 1:1, most likely by interacting with the hydration shell. Here by DSF and NMR, we characterized the effect of ATP on binding, conformation, stability of G18V γS-crystallin and its interactions with α-crystallin. The results reveal: 1) G18V significantly accelerates the crowding-induced destabilization with Tm of 67 °C reduced to 50.5 °C at 1 mM. 2) Most unexpectedly, G18V almost completely eliminates the antagonizing effect of ATP against the crowding-induced destabilization. 3) ATP shows no significant effect on the interactions of α-crystallin with both WT and G18V γS-crystallin. Results together decode for the first time that G18V causes cataract not only by accelerating the crowding-induced destabilization, but also by eliminating the antagonizing effect of ATP against the crowding-induced destabilization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

One of the most crowded biological environments is the eye lens which contains a high concentration of crystallin proteins. The molecular chaperones αB-crystallin (αBc) with its lens partner αA-crystallin (αAc) prevent deleterious crystallin aggregation and cataract formation. However, some forms of cataract are associated with structural alteration and dysfunction of αBc. While many studies have investigated the structure and function of αBc under dilute in vitro conditions, the effect of crowding on these aspects is not well understood despite its in vivo relevance. The structure and chaperone ability of αBc under conditions that mimic the crowded lens environment were investigated using the polysaccharide Ficoll 400 and bovine γ-crystallin as crowding agents and a variety of biophysical methods, principally contrast variation small-angle neutron scattering. Under crowding conditions, αBc unfolds, increases its size/oligomeric state, decreases its thermal stability and chaperone ability, and forms kinetically distinct amorphous and fibrillar aggregates. However, the presence of αAc stabilizes αBc against aggregation. These observations provide a rationale, at the molecular level, for the aggregation of αBc in the crowded lens, a process that exhibits structural and functional similarities to the aggregation of cataract-associated αBc mutants R120G and D109A under dilute conditions. Strategies that maintain or restore αBc stability, as αAc does, may provide therapeutic avenues for the treatment of cataract., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 41 GCWMLY 46 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In lens, αβγ-crystallins accounting for ∼90% of ocular proteins with concentrations >400 mg/ml need to remain soluble for the whole life-span and their aggregation can lead to cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3-7 mM. Very recently, ATP was proposed to hydrotropically prevent aggregation of crystallins but the mechanism remains unexplored. Here by NMR, DLS and DSF, we characterized the association, thermal stability and conformation of the 178-residue human γS-crystallin at concentrations from 2 to 100 mg/ml in the absence and in the presence of ATP. Results together reveal for the first time that ATP does antagonize the crowding-induced destabilization, although it has no significant binding to γS-crystallin as well as no alteration of its conformation. Therefore, ATP prevents aggregation in lens by a novel mechanism, thus rationalizing the fact that declining concentrations of ATP upon being aged is related to age-related cataractogenesis. To restore the normal concentrations of ATP in lens may represent a promising therapeutic strategy to treat aggregation-causing eye diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity., Methods: Molecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A., Results: γS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature., Conclusions: αB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant., General Significance: Human αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 (10 3 - 10 4  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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1 H N chemical shifts using solution NMR spectroscopy. Consistent with the Ca 2+ -binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oxidative aggregation of γ-crystallins induced by copper in aged lens increases the lens opacity and causes cataract formation. Therefore, chelation of free Cu 2+ 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 Cu 2+ 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 ~10 5  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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The reducing environment in the eye lens diminishes with age, leading to significant oxidative stress. Oxidation of lens crystallin proteins is the major contributor to their destabilization and deleterious aggregation that scatters visible light, obscures vision, and ultimately leads to cataract. However, the molecular basis for oxidation-induced aggregation is unknown. Using X-ray crystallography and small-angle X-ray scattering, we describe the structure of a disulfide-linked dimer of human γS-crystallin that was obtained via oxidation of C24. The γS-crystallin dimer is stable at glutathione concentrations comparable to those in aged and cataractous lenses. Moreover, dimerization of γS-crystallin significantly increases the protein's propensity to form large insoluble aggregates owing to non-cooperative domain unfolding, as is observed in crystallin variants associated with early-onset cataract. These findings provide insight into how oxidative modification of crystallins contributes to cataract and imply that early-onset and age-related forms of the disease share comparable development pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Liquid-liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein-protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human γS-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human γS-crystallin, Y67N (abbreviated hereafter as γS-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for γS-Y67N to cause lens opacification, we constructed, expressed and purified γS-Y67N and its wild-type (abbreviated hereafter as γS-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of γS-crystallin upon mutation associated with enhanced aggregation. As Ca 2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca 2+ -binding properties of γS-WT and γS-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca 2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu 2+ -induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cataract, a crystallin protein aggregation disease, is the leading cause of human blindness worldwide. Congenital cataract may be induced by many factors and genetic disorders accounts for about half of the cases. Inherited mutations can promote cataract formation by affecting crystallin structure, solubility, stability, protein interactions and aggregatory propensity. In this research, we investigated the potential role of oxidative damage in congenital cataracts caused by six mutations in γC- and γD-crystallins, the predominant structural proteins in the lens. H 2 O 2 treatment induced structural changes for both the wild type and mutated proteins. Oxidization by H 2 O 2 or UV light facilitated protein oligomerization and thermal aggregation. H 2 O 2 treatment promoted thermal aggregation of all proteins. By increasing the susceptibility, cataract-causing mutations amplified the deleterious effects of oxidative damage. Our results suggested that oxidative damage might play an important role in the onset and/or progression of congenital cataract caused by both Cys and non-Cys substitutions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glycation of ocular lens proteins plays a vital role in the development of diabetic cataract. In order to investigate the role of glycation in cataractogenesis, the extent of glycation of human γB-crystallin was determined by an in vitro glycation study in a solution of high glucose content for upto 28days. The glycated protein has been purified and the formation of advanced glycation end products (AGEs) has been monitored spectroscopically. Size exclusion chromatographic studies showed that the covalent intermolecular crosslinking in the dimer formed was not due to disulfide bond formation. MALDI-TOF spectroscopy was employed to determine the number of glucose moieties attached to the protein due to glycation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Disruption of the short range order of proteins present in the ocular lens leads to cataract resulting in a loss of transparency. Human γ-crystallin (HGC), a water soluble protein present in the lens is known to aggregate with aging. A modified form of HGC (HGC(c)) was isolated from cataractous human ocular lens extract and the number of Trp residues that undergo oxidation was determined. The extent of oxidized Trp (N-formyl kynurenine) in HGC due to cataract formation was determined, primarily using fluorescence spectroscopy. The ability of (-)-epigallocatechin gallate (EGCG) to retain its antioxidant effect even in the presence of H2O2 was investigated. This was monitored by its ability to prevent the modification of intact Trp residues in HGC(c) isolated from cataractous human eye lens. Significant Trp fluorescence quenching occurs on interaction of the green tea component, EGCG with HGC(c) accompanied by a red shift. Docking studies were employed to substantiate the experimental results. As eye lens proteins are prone to oxidative stress it is essential that a clear understanding of the effects of the components generated in vivo vis-à-vis the antioxidant effects of natural polyphenols be obtained., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A number of point mutations in γD-crystallin are associated with human cataract. The Pro23-to-Thr (P23T) mutation is perhaps the most common, is geographically widespread, and presents itself in a variety of phenotypes. It is therefore important to understand the molecular basis of lens opacity due to this mutation. In our earlier studies, we noted that P23T shows retrograde and sharply lowered solubility, most likely due to the emergence of hydrophobic patches involved in protein aggregation. Binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) dye (a probe commonly used for detecting surface hydrophobicity) competed with aggregation, suggesting that the residues involved in Bis-ANS binding are also involved in protein aggregation. Here, using NMR spectroscopy in conjunction with Bis-ANS binding, we identify three residues (Y16, D21, and Y50) in P23T that are involved in binding the dye. Furthermore, using (15)N NMR relaxation experiments, we show that, in the mutant protein, backbone fluctuations are restricted to the picosecond-to-nanosecond and microsecond timescales relative to the wild type. Our present studies specify the residues involved in these two pivotal characteristics of the mutant protein, namely increased surface hydrophobicity and restricted mobility of the protein backbone, which can explain the nucleation and further propagation of protein aggregates. Thus, we have now identified the residues in the P23T mutant that give rise to novel hydrophobic surfaces, as well as those regions of the protein backbone where fluctuations in different timescales are restricted, providing a comprehensive understanding of how lens opacity could result from this mutation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Crystallin is essential not only for the maintenance of eye lens transparency, but also in the biology of other tissues. Eye lens α-crystallin exists as a heteropolymer composed of two homologous subunits, αA and αB. Despite the critical role of α-crystallin in many tissues, little is known regarding structural and functional significance of the two subunits. Herein, we describe a unique feature of αB-crystallin. At high temperatures (>70°C) not only αB-crystallin aggregates but also enhances the aggregation of other lens proteins. Intriguingly, αB-crystallin-mediated coaggregation at and above 70°C involves β- but not γ-crystallin. Further, αA-crystallin, but not a mutant (F71L) αA-crystallin, prevented aggregation of αB-crystallin and also reduced coaggregation of αB- and β-crystallin. These studies explain the rationale for the existence of α-crystallin heteropolymer with αA subunit as a major partner that is vital for lens transparency and provide insights into αB-crystallin-induced coaggregation which may have a bearing in some pathological conditions where αB-crystallin is overexpressed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-crystallin oligomers formed long-lived stable complexes with their gammaD-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with alpha-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The lens capsule compartmentalizes the cells of the avascular lens from other ocular tissues. Small molecules required for lens cell metabolism, such as glucose, salts, and waste products, freely pass through the capsule. However, the lens capsule is selectively permeable to proteins such as growth hormones and substrate carriers which are required for proper lens growth and development. We used fluorescence recovery after photobleaching (FRAP) to characterize the diffusional behavior of various sized dextrans (3, 10, 40, 150, and 250 kDa) and proteins endogenous to the lens environment (EGF, gammaD-crystallin, BSA, transferrin, ceruloplasmin, and IgG) within the capsules of whole living lenses. We found that proteins had dramatically different diffusion and partition coefficients as well as capsule matrix binding affinities than similar sized dextrans, but they had comparable permeabilities. We also found ionic interactions between proteins and the capsule matrix significantly influence permeability and binding affinity, while hydrophobic interactions had less of an effect. The removal of a single anionic residue from the surface of a protein, gammaD-crystallin [E107A], significantly altered its permeability and matrix binding affinity in the capsule. Our data indicated that permeabilities and binding affinities in the lens capsule varied between individual proteins and cannot be predicted by isoelectric points or molecular size alone., (Copyright 2009 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Diminished proteolytic functionality in the lens may cause cataracts. We have reported that O-GlcNAc is an endogenous inhibitor of the proteasome. We hypothesize that in the lens there is a cause-and-effect relationship between proteasome inhibition by O-GlcNAc, and cataract formation. To demonstrate this, we established novel transgenic mouse models to over-express a dominant-negative form of O-GlcNAcase, GK-NCOAT, in the lens. Expression of GK-NCOAT suppresses removal of O-GlcNAc from proteins, resulting in increased levels of O-GlcNAc in the lenses of our transgenic mice, along with decreased proteasome function. We observed that transgenic mice developed markedly larger cataracts than controls and lens fiber cell denucleation was inhibited. Our study suggests that increased O-GlcNAc in the lens could lead to cataract formation and attenuation of lens fiber cell denucleation by inhibition of proteasome function. These findings may explain why cataract formation is a common complication of diabetes since O-GlcNAc is derived from glucose.

Pitx3 is a bicoid like homeobox transcription factor of which deficiency in mice is linked with the aphakia phenotype. Mutation in human PITX3 gene is associated with autosomal dominant cataract with variable anterior segment mesenchymal dysgenesis. However, the molecular events causing the morphological changes in aphakia remains unknown. In this study we investigated the behaviour of GFP tagged Pitx3 null embryonic stem cells in chimeric lens, as well as the molecular features of the Pitx3-deficient lens of homozygous Pitx3 knockout mice. We show that the lack of colonisation of Pitx3-deficient ES cell derivatives in Pitx3 wild-type<-->Pitx3 null chimeric lens was due to the depletion of the epithelial cells in lens epithelium manifested by aberrant cell cycle exit and precocious onset of fibre cell differentiation of the Pitx3 null cells at the lens vesicle stage. This was demonstrated by the early activation of the cell cycle inhibitors p27Kip1 and p57Kip2, and the expression of beta-and gamma-crystallins. These defects are at least partially attributed to the loss of FoxE3 and misexpression of Prox1 in the lens vesicle epithelial cells. Thus, Pitx3 is essential to maintain lens epithelial phenotype and prevent inappropriate fibre cell differentiation during lens development.

Essential and Molecular Dynamics (ED/MD) have been used to model the conformational changes of a protein implicated in a conformational disease--cataract, the largest cause of blindness in the world-after non-enzymic post-translational modification. Cyanate modification did not significantly alter flexibility, while the Schiff's base adduct produced a more flexible N-terminal domain, and intra-secondary structure regions, than either the cyanate adduct or the native structure. Glycation also increased linker flexibility and disrupted the charge network. A number of post-translational adducts showed structural disruption around Cys15 and increased linker flexibility; this may be important in subsequent protein aggregation. Our modelling results are in accord with experimental evidence, and show that ED/MD is a useful tool in modelling conformational changes in proteins implicated in disease processes.

The structural and functional characteristics of a yeast alcohol dehydrogenase (ADH) peptide (YSGVCHTDLHAWHGDWPLPVK, residues 40-60) have been studied in detail. The peptide is hydrophobic in nature, binds the hydrophobic probe bis-ANS, and is mostly present in a random coil conformation. It shows chaperone-like activity by preventing dithiothreitol (DTT)-induced aggregation of insulin at 27 degrees C, oxidation-induced aggregation of gamma-crystallin at 37 degrees C, and aggregation of thermally denatured ADH and beta(L)-crystallins at 52 degrees C. However, the ADH peptide does not solubilize protein aggregates as do surfactants. Substitution of Pro for His in the ADH peptide leads to diminished anti-aggregation activity. Further, analysis of ADH incubated at 47 degrees C suggests that a significant portion of the enzyme remains as soluble inactive protein with negligible conformational change. Therefore, we propose that the residues 40-60 in native protein may be an intramolecular chaperone site of yeast ADH.

Gamma crystallin is one of three structural proteins present in great abundance in the fiber cells of the vertebrate eye lens. The protein displays a tendency to aggregate readily in the course of heating, cooling, being exposed to ultraviolet radiation, or rapid refolding. To investigate the molecular mechanisms underlying such aggregation, we have employed a peptide-scanning approach aimed at identifying regions of bovine gamma-II crystallin that may be involved in intermolecular interactions leading to aggregation, using assays that measure the competitive inhibition of such aggregation by reagents drawn from a group of contiguous (overlapping) peptides derived from the sequence of the protein itself. Our results suggest that two regions, comprising residues 61-74, and 145-159, play key roles in aggregative interactions. Intriguingly, the two regions (each containing a solvent-exposed, single-turn helix in the native structure) are located in structurally analogous positions in the two homologous double Greek key (beta sheet) domains of the protein, suggesting that helix-strand conversions may operate to facilitate intermolecular beta sheet interactions during aggregation.