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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.)

Cataract is one of the leading causes of blindness worldwide. Till date, the only solution for cataracts is surgery, which is a resource-intensive solution. A much simpler solution is to find a potential drug that could inhibit aggregation. It is well established that nonamyloid aggregates of eye lens protein result in cataract. γD-Crystallin, a thermodynamically stable protein, is one of the most abundant proteins in the core of the eye lens and is found to aggregate under stress conditions, leading to the cataract. It has also been found that in cataractous lens, the concentration of metals like copper is elevated significantly as compared to healthy eye lens, suggesting their role in inducing aggregation. In our present study, aggregation of γD-Crystallin was carried out in the presence of Cu (II). Using techniques like turbidity assay, CD spectroscopy, ANS binding assay, and microscopic studies like TEM, it could be confirmed that protein aggregates in the presence of Cu (II) and the nature of aggregates is amorphous. Various polyphenols were tested to suppress aggregation of the protein. Quercetin was observed to be the most efficient. To overcome the problems associated with the delivery of polyphenols, such as solubility and bioavailability, quercetin was encapsulated in two types of nanocarriers. Their characterization was done using TEM, DLS, and other techniques. The potency of quercetin-loaded CS-TPP/CS-PLGA NPs as inhibitors of γD-Crystallin aggregation was confirmed by various experiments.

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 1 H- 15 N 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., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Spontaneous deamidation of amino acids is a physiologically important process, particularly for protein aging and diseases. Despite its widespread occurrence, the mechanism of glutamine deamidation particularly within proteins remains poorly understood. We have used a multiscale computational approach to investigate glutamine deamidation in the tripeptide Glycine-Glutamine-Glycine (Gly-Gln-Gly) and γS-Crystallin protein. Specifically, both the 5- and 6-membered water-assisted deamidation pathways in the tripeptide have been elucidated and compared. Both are found to occur in three stages: iminol formation, cyclization, and deamination. The rate-limiting step in each mechanism is nucleophilic attack of the backbone iminol nitrogen, formed in the first stage, at the glutamine's side-chain carbonyl carbon. For the 6- and 5-membered mechanisms, this occurs with a free energy cost of 136.4 and 179.5 kJ mol -1 , respectively. Thus, overall, in the Gly-Gln-Gly tripeptide, the 6-membered pathway is preferred. Furthermore, the free energies for forming cyclic intermediates and products at selected Gln residues (based on experimentally reported % deamidation) in γS-Crystallin have been obtained. It is found that the 5-membered product complex is exergonic at -25.3 kJ mol -1 , while the 6-membered product complex is calculated to be endergonic at 90.7 kJ mol -1 . Thus, the deamidation pathway in folded and constrained proteins may not exclusively follow the 6-membered route. Molecular dynamics (MD) simulations of γS-Crystallin indicate that deamidation is more likely to occur when two or more water molecules are in the proximity of the glutamine residue. Consequently, significant conformational changes are found to accompany Gln120 deamidation in γS-Crystallin. This in turn can influence water availability at the other Gln residues considered and hence potentially their deamidation. Collectively, these results provide comprehensive insights into spontaneous water-assisted deamidation of glutamine residues in peptides and into the role and impact of Gln deamidation in proteins.

Human γD-crystallin belongs to a crucial family of proteins known as crystallins located in the fiber cells of the human lens. Since crystallins do not undergo any turnover after birth, they need to possess remarkable thermodynamic stability. However, their sporadic misfolding and aggregation, triggered by environmental perturbations or genetic mutations, constitute the molecular basis of cataracts, which is the primary cause of blindness in the globe according to the World Health Organization. Here, we investigate the impact of high pressure on the conformational landscape of wild-type HγD-crystallin using replica exchange molecular dynamics simulations augmented with principal component analysis. We find pressure to have a modest impact on global measures of protein stability, such as root-mean-square displacement and radius of gyration. Upon projecting our trajectories along the first two principal components from principal component analysis, however, we observe the emergence of distinct free energy basins at high pressures. By screening local order parameters previously shown or hypothesized as markers of HγD-crystallin stability, we establish correlations between a tyrosine-tyrosine aromatic contact within the N-terminal domain and the protein's end-to-end distance with projections along the first and second principal components, respectively. Furthermore, we observe the simultaneous contraction of the hydrophobic core and its intrusion by water molecules. This exploration sheds light on the intricate responses of HγD-crystallin to elevated pressures, offering insights into potential mechanisms underlying its stability and susceptibility to environmental perturbations, crucial for understanding cataract formation.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and βγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the βγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens βγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in βB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even β-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6 + , GJA1 - , β-crystallin - and γ-crystallin - cells; the germinative zone, characterized by PAX6 + , GJA1 + , β-crystallin - and γ-crystallin - ; the transitional zone, characterized by PAX6 + , GJA1 + , β-crystallin + and γ-crystallin - ; and the equatorial zone, characterized by PAX6 +/- , GJA1 + , β-crystallin + , and γ-crystallin + cells. The putative lens epithelial stem cells identified as SOX2 + and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

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.)

Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The β L - and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of β L - and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that β L -crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when β L -crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when β L - and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both β L - and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when β L -crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of β L - and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.

The molecular basis underlying the rich phase behavior of globular proteins remains poorly understood. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics and interprotein interactions of γ D-crystallin and its P23T-R36S mutant, which drastically limits the protein solubility, at both infinite dilution and at a concentration where the mutant fluid phase and crystalline phase coexist. We find that while the mutant conserves the protein fold, changes to the surface exposure of residues in the neighborhood of residue-36 enhance protein-protein interactions and develop specific protein-protein contacts found in the protein crystal lattice., (© 2023 Wiley Periodicals LLC.)

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, β-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

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.)

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H 2 O 2  and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved T m  of both H 2 O 2  oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins., Competing Interests: Conflict of interest V. M. M. is on the Scientific Advisory Board of Revel Pharmaceuticals. Inc. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

As part of the development of an infectious bursal disease virus (IBDV) subunit vaccine, this study was designed to improve the expression of highly soluble VP2-LS3 (Haemophilus parasuis lumazine synthase 3, LS3) protein by using different tagged vectors in E. coli . IBDV VP2-LS3 gene was designed and synthesized. Fusion tags, GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin were joined to the N-terminus of VP2-LS3 protein. Seven expression plasmids were constructed, and each plasmid was transformed into E. coli BL21 (DE3) competent cells. After induction by IPTG, the solubility and expression levels of the various VP2-LS3 proteins were analyzed by SDS-PAGE and Western Blot analysis. The fusion tag that significantly promoted soluble expression of the VP2-LS3 protein was selected. Recombinant proteins were purified using Ni-NTA affinity chromatography, then cleaved by using TEV protease and detected by using transmission electron microscopy. Gel electrophoresis and sequencing analysis showed that all seven recombinant vectors were successfully constructed. GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin enhanced the expression and solubility of VP2 protein; however, MBP was more effective for the high-purity production of VP2-LS3. Western Blot analysis confirmed successful generation of VP2-LS3 fusion protein in E. coli . The result of transmission electron microscopy showed that VP2-LS3 formed nano-sized particles with homogeneous shape and relatively uniform size. This study established a method to generate VP2-LS3 recombinant protein, which may lay a foundation for the development and subsequent study of IBDV subunit vaccines.

Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γS-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference., Competing Interests: The authors have declared that no competing interests exist.

The nuclear region of the lens is metabolically quiescent, but it is far from inert chemically. Without cellular renewal and with decades of environmental exposures, the lens proteome, lipidome, and metabolome change. The lens crystallins have evolved exquisite mechanisms for resisting, slowing, adapting to, and perhaps even harnessing the effects of these cumulative chemical modifications to minimize the amount of light-scattering aggregation in the lens over a lifetime. Redox chemistry is a major factor in these damages and mitigating adaptations, and as such, it is likely to be a key component of any successful therapeutic strategy for preserving or rescuing lens transparency, and perhaps flexibility, during aging. Protein redox chemistry is typically mediated by Cys residues. This review will therefore focus primarily on the Cys-rich γ-crystallins of the human lens, taking care to extend these findings to the β- and α-crystallins where pertinent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of β- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. 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.)

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.

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.)

Objective: This study aimed to identify the disease-causing mutation of congenital cataract disease in a large northeastern Chinese family., Materials and Methods: The subjects' peripheral blood was collected, their genomic DNA was extracted, mutation screening of candidate genes was performed using polymerase chain reaction, and the amplified products were sequenced. Recombinant C-terminal enhanced green fluorescent protein-tagged wild-type or mutant CRYGD was expressed in HEK293T cells, and the expression pattern was observed under a fluorescence microscope. The CRYGD protein mutation was analyzed via bioinformatics analysis., Results: c.475delG, a novel frameshift mutation in CRYGD, was identified in the affected family members. This mutation causes premature termination of the polypeptide, resulting in truncated p.(Ala159ProfsTer9). According to the bioinformatics analysis results, compared with wild-type CRYGD, p.(Ala159ProfsTer9) exhibits significantly decreased hydrophilicity. Fluorescence microscopy revealed that p.(Ala159ProfsTer9) aggregates in the cell in the form of granular deposits., Conclusion: In this study, the novel frameshift mutation c.475delG, p.(Ala159ProfsTer9) in CRYGD was identified to cause congenital cataracts in a large Chinese family; increased hydrophobicity of p.(Ala159ProfsTer9) protein may be the underlying mechanism., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

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.)

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

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.)

β/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 β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-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 β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

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.)

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, βH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies., Competing Interests: The authors have declared that no competing interests exist.

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.)

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia., (© 2020 Nandi et al.)

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas β-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS., (© 2019 John Wiley & Sons, Ltd.)

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.)

Oligomerization and aggregation of γD-crystallins (HγDC) in the eye lens is one of the main causes of cataract development. To date, several congenital mutations related to this protein are known to promote the formation of aggregates. Previous studies have demonstrated that mutations in W42 residue of HγDC lead to the generation of partially unfolded intermediates that are more prone to aggregate. To understand the role of W42 in the stability of HγDC, we performed alchemical free-energy calculations and all-atom molecular dynamics simulations of different W42 mutant models. Our results suggest that substitution of W42 by small size and/or polar residues promotes HγDC denaturation due to the entry of water molecules into the hydrophobic core of the N-terminal domain. Similar behavior was observed in the C-terminal domain of HγDC when mutating the W130 residue located in a homologous position. Moreover, the exposure of the hydrophobic core residues could lead to the formation of aggregation-prone partially unfolded species. Overall, this study takes a step toward understanding the role of HγDC in cataract development.

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.)

The βγ -crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ -crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ -crystallins bind calcium ions. The βγ -crystallin from the tunicate Ciona intestinalis ( Ci - βγ ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci - βγ is valuable for investigating the evolution of the βγ -crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca 2+ and other divalent cations on the stability and aggregation propensity of Ci - βγ and human γS-crystallin (HγS). Beyond Ca 2+ , Ci - βγ is capable of coordinating Mg 2+ , Sr 2+ , Co 2+ , Mn 2+ , Ni 2+ , and Zn 2+ , although only Sr 2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci - βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn 2+ , Ni 2+ , and Co 2+ induce aggregation by interacting with cysteine residues, whereas Cu 2+ -mediated aggregation proceeds via a different binding site.

The prokaryotic βγ-crystallins are a large group of uncharacterized domains with Ca 2+ -binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca 2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii , a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a βγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, β-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca 2+ -binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the β-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca 2+ binding and comparing different domain combinations, we demonstrate that the βγ-crystallin domain in glucanallin acts as a Ca 2+ sensor and enhances the glycolytic activity of glucanallin through Ca 2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members. IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca 2+ via assistance from a neighboring βγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a "βγ-crystallination"-mediated Ca 2+ sensitivity may be widespread among bacterial proteins., (Copyright © 2019 American Society for Microbiology.)

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro . However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro . We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15 N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

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.)

The mechanisms leading to aggregation of the crystallin proteins of the eye lens remain largely unknown. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics of γD-crystallin and its cataract-related W42R variant at both infinite dilution and physiologically relevant concentrations. We find that the W42R variant assumes a distinct conformation in solution that leaves the Greek key domains of the native fold largely unaltered but lacks the hydrophobic interdomain interface that is key to the stability of wild-type γD-crystallin. At physiologically relevant concentrations, exposed hydrophobic regions in this alternative conformation become primary sites for enhanced interprotein interactions leading to large-scale aggregation.

The γ-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. γD- and γS-crystallin are two major monomeric crystallins of the human lens. γD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas γS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins' isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability., (Copyright © 2019 Biophysical Society. Published by 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.)

A recently identified mutant of human γS-crystallin, G57W is associated with dominant congenital cataracts, the familial determinate of childhood blindness worldwide. To investigate the structural and functional changes that mediate the effect of this cataract-related mutant to compromise eye lens transparency and cause lens opacification in children, we recently reported complete sequence-specific resonance assignments of γS-G57W using a suite of heteronuclear NMR experiments. As a follow up, we have determined the 3D structure of γS-G57W and studied its conformational dynamics by solution NMR spectroscopy. Our structural dynamics results reveal greater flexibility of the N-terminal domain, which undergoes site-specific structural changes to accommodate W57, than its C-terminal counterpart. Our structural inferences that the unusual solvent exposure of W57 is associated with rearrangement of the N-terminal domain suggest an efficient pathway for increased aggregation in γS-G57W and illuminates the molecular dynamics underlying cataractogenic aggregation of lens crystallins in particular and aggregation of proteins in general., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid-liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun. 2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x ). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer-dimer self-association with K D = 24 mM, corresponding to a standard free energy change of approximately -9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups., Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 μmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30., Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, β-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, β-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting., Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

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.)