Interaction of ßL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy.

Highly concentrated lens proteins, mostly ß- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, ß- 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 ß- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of ß- 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 ß- 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 ßL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of ßL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that ßL-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 ßL-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 ßL- 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 ßL- 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 ßL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of ßL- 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.

Oxidation of active cysteines mediates protein aggregation of S10R, the cataract-associated mutant of mouse GammaB-crystallin.

The Ser10 to Arg mutation in mouse γB-crystallin (MGB) has been associated with protein aggregation, dense nuclear opacity, and the degeneration of fiber cells in the lens core. Overexpression of the gap junction protein, connexin 46 (Cx46), was found to suppress the nuclear opacity and restore normal cell-cell contact. However, the molecular basis for the protein aggregation and related downstream effects were not evident from these studies. Here, we provide a comparison of the structures and solution properties of wild type MGB and the S10R mutant in vitro and show that, even though the mutation does not directly involve cysteine residues, some cysteines in the mutant protein are activated, leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates relative to the wild-type. This occurs even as the protein structure is essentially unaltered. Thus, the primary event is enhanced protein aggregation due to the disulfide crosslinking of the mutant protein. We suggest that these aggregates eventually get deposited on fiber cell membranes. Since the gap junction protein, Cx46 is involved in the transport of reduced glutathione, we posit that these deposits interfere in Cx46-mediated glutathione transport and facilitate the oxidative stress-mediated downstream changes. Overexpression of Cx46 suppresses such oxidative aggregation. These studies provide a plausible explanation for the protein aggregation and other changes that accompany this mutation. If indeed cysteine oxidation is the primary event for protein aggregation also in vivo, then the S10R mutant mouse, which is currently available, could serve as a viable animal model for human age-onset cataract.

Crystallins and Their Complexes.

The crystallins (α, ß and γ), major constituent proteins of eye lens fiber cells play their critical role in maintaining the transparency and refractive index of the lens. Under different stress factors and with aging, ß- and γ-crystallins start to unfold partially leading to their aggregation. Protein aggregation in lens basically enhances light scattering and causes the vision problem, commonly known as cataract. α-crystallin as a molecular chaperone forms complexes with its substrates (ß- and γ-crystallins) to prevent such aggregation. In this chapter, the structural features of ß- and γ-crystallins have been discussed. Detailed structural information linked with the high stability of γC-, γD- and γS-crystallins have been incorporated. The nature of homologous and heterologous interactions among crystallins has been deciphered, which are involved in their molecular association and complex formation.

Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases.

Protein aggregation, due to the imbalance in the concentration of Cu2+ and Zn2+ ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of ß/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule 'chelator' for Cu2+ with complementary antioxidant effect will find potential applications against aggregation of ß/γ-crystallins. In this paper, we have reported the synthesis of different Schiff bases and studied their Cu2+ complexation ability (using UV-Vis, FT-IR and ESI-MS) and antioxidant activity. Further based on their copper complexation efficiency, Schiff bases were used to inhibit Cu2+-mediated aggregation of recombinant human γD-crystallin (HGD) and ß/γ-crystallins (isolated from cataractous human eye lens). Among these synthesized molecules, compound 8 at a concentration of 100 µM had shown ~95% inhibition of copper (100 µM)-induced aggregation. Compound 8 also showed a positive cooperative effect at a concentration of 5-15 µM on the inhibitory activity of human αA-crystallin (HAA) during Cu2+-induced aggregation of HGD. It eventually inhibited the aggregation process by additional ~20%. However, ~50% inhibition of copper-mediated aggregation of ß/γ-crystallins (isolated from cataractous human eye lens) was recorded by compound 8 (100 µM). Although the reductive aminated products of the imines showed better antioxidant activity due to their lower copper complexing ability, they were found to be non-effective against Cu2+-mediated aggregation of HGD.

Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation.

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.

Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: an implications for anti-cataract agent.

Coumarin derivative 1, 5,7-dihydroxy-6-(3-methyl-1-butyryl)-4-phenyl-chromen- 2-one, has been reported to possess radical scavenging activity and DNA protection. We have synthesized a series of coumarins with structural modifications at positions C4, C5, C6 and C7 and evaluated them for their anti-UVC properties. Coumarin 7, 6-benzoyl-5,6-dihydroxy-4-phenyl-chromen-2-one, was found to have the most potent activity in protecting porcine γ-crystallin against UVC insults. Results of fluorescence assays indicated that compound 7 was capable of decreasing the loss of intensity while lens crystallins and DNA PUC19 were irradiated with UVC. Presence of compound 7 decreased hydroxyl radical levels determined by probe 1b and the free iron concentrations determined by Ferrozine reagent. The chelation assay showed that compound 7 was chelated to metal via 6-CO and 5-OH on the benzopyrone ring. The observed protective effects of compound 7 towards crystallins from insults of UVC and free radicals may be due to its iron-chelating activity and its peak absorption at 254 nm.

Increasing susceptibility to oxidative stress by cataract-causing crystallin mutations.

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. H2O2 treatment induced structural changes for both the wild type and mutated proteins. Oxidization by H2O2 or UV light facilitated protein oligomerization and thermal aggregation. H2O2 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.

Cataract-causing mutation S228P promotes ßB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain.

ß/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various ß/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in ßB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of ßB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native ßB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of ßB1-crystallin on ßA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native ßB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all ß/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of ß/γ-crystallin domains by protecting the hydrophobic core from solvent access.

EGCG prevents tryptophan oxidation of cataractous ocular lens human γ-crystallin in presence of H2O2.

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.