Manipulating polydispersity of lens ß-crystallins using divalent cations demonstrates evidence of calcium regulation.

Crystallins comprise the protein-rich tissue of the eye lens. Of the three most common vertebrate subtypes, ß-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the ß-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized ß-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl2 or MgCl2). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate ß-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge-properties that can be reversed when salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms of ß-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.

Binding of ßL-Crystallin with Models of Animal and Human Eye Lens-Lipid Membrane.

Several discoveries show that with age and cataract formation, ß-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence ß-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of ß-crystallin to the membrane and the membrane's physical properties (mobility, order, and hydrophobicity) with ß-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of ßL-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that ßL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by ßL-crystallin and the binding affinity (Ka) of ßL-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and Ka (MHLL) > Ka (MMLL) ≈ Ka (MPLL), respectively, in which the presence of Chol reduces the MMSO and Ka for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of ßL-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in ßL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with ßL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that ßL-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with ßL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

Zinc and Copper Ions Induce Aggregation of Human ß-Crystallins.

Cataracts are defined as the clouding of the lens due to the formation of insoluble protein aggregates. Metal ions exposure has been recognized as a risk factor in the cataract formation process. The γ and ß crystallins are members of a larger family and share several structural features. Several studies have shown that copper and zinc ions induce the formation of γ-crystallins aggregates. However, the interaction of metal ions with ß-crystallins, some of the most abundant crystallins in the lens, has not been explored until now. Here, we evaluate the effect of Cu(II) and Zn(II) ions on the aggregation of HßA1, as a representative of the acidic form, and HßB2, as a representative of the basic ß-crystallins. We used several biophysical techniques and computational methods to show that Cu(II) and Zn(II) induce aggregation following different pathways. Both metal ions destabilize the proteins and impact protein folding. Copper induced a small conformational change in HßA1, leading to high-molecular-weight light-scattering aggregates, while zinc is more aggressive towards HßB2 and induces a larger conformational change. Our work provides information on the mechanisms of metal-induced aggregation of ß-crystallins.

Crowding in the Eye Lens: Modeling the Multisubunit Protein ß-Crystallin with a Colloidal Approach.

We present a multiscale characterization of aqueous solutions of the bovine eye lens protein ßH crystallin from dilute conditions up to dynamical arrest, combining dynamic light scattering, small-angle x-ray scattering, tracer-based microrheology, and neutron spin echo spectroscopy. We obtain a comprehensive explanation of the observed experimental signatures from a model of polydisperse hard spheres with additional weak attraction. In particular, the model predictions quantitatively describe the multiscale dynamical results from microscopic nanometer cage diffusion over mesoscopic micrometer gradient diffusion up to macroscopic viscosity. Based on a comparative discussion with results from other crystallin proteins, we suggest an interesting common pathway for dynamical arrest in all crystallin proteins, with potential implications for the understanding of crowding effects in the eye lens.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure.

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.

ßγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation.

The prokaryotic ßγ-crystallins are a large group of uncharacterized domains with Ca2+-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 Ca2+ 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 ßγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, ß-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the ß-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 Ca2+ binding and comparing different domain combinations, we demonstrate that the ßγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ 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 Ca2+ via assistance from a neighboring ßγ-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 "ßγ-crystallination"-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

Dissimilarity in the Contributions of the N-Terminal Domain Hydrophobic Core to the Structural Stability of Lens ß/γ-Crystallins.

Vertebrate lens ß/γ-crystallins share a conserved tertiary structure consisting of four Greek-key motifs divided into two globular domains. Numerous inherited mutations in ß/γ-crystallins have been linked to cataractogenesis. In this research, the folding mechanism underlying cataracts caused by the I21N mutation in ßB2 was investigated by comparing the effect of mutagenesis on the structural features and stability of four ß/γ-crystallins, ßB1, ßB2, γC, and γD. Our results showed that the four ß/γ-crystallins differ greatly in solubility and stability against various stresses. The I21N mutation greatly impaired ßB2 solubility and native structure as well as its stability against denaturation induced by guanidine hydrochloride, heat treatment, and ultraviolet irradiation. However, the deleterious effects were much weaker for mutations at the corresponding sites in ßB1, γC, and γD. Molecular dynamics simulations indicated that the introduction of a nonnative hydrogen bond contributed to twisting Greek-key motif I outward, which might direct the misfolding of the I21N mutant of ßB2. Meanwhile, partial hydration of the hydrophobic interior of the domain induced by the mutation destabilized ßB1, γC, and γD. Our findings highlight the importance of nonnative hydrogen bond formation and hydrophobic core hydration in crystallin misfolding caused by inherited mutations.

Divalent Cations and the Divergence of ßγ-Crystallin Function.

The ßγ-crystallin superfamily contains both ß- 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 ßγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial ßγ-crystallins bind calcium ions. The ßγ-crystallin from the tunicate Ciona intestinalis (Ci-ßγ) 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-ßγ is valuable for investigating the evolution of the ßγ-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 Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-ßγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-ßγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-ßγ 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. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

Interface interactions between ßγ-crystallin domain and Ig-like domain render Ca2+ -binding site inoperative in abundant perithecial protein of Neurospora crassa.

We describe a set of proteins in which a ßγ-crystallin domain pairs with an Ig-like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD-1 (Ca2+ -dependent cell adhesion molecule-1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP-NTD (N-terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+ -binding site within this class of molecules. As a common feature, one of the two Ca2+ -binding sites is non-functional in the ßγ-crystallin domains of these proteins. While APP-NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD-1, APP surprisingly does not bind Ca2+ . Crystal structures of APP and Ca2+ -bound APP-NTD reveal that the interface interactions in APP render its Ca2+ -binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+ -binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig-like domain removes the constraints upon the required conformational transition and enables the ßγ-crystallin domain to bind Ca2+ . In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD-1.

Polyphenol-enriched fraction of Vaccinium uliginosum L. protects selenite-induced cataract formation in the lens of Sprague-Dawley rat pups.

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-, ß-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, ß-, 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.

Biochemical characterization of G64W mutant of acidic beta-crystallin 4.

Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic ß-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic ß-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.

Endogenous bioelectric currents promote differentiation of the mammalian lens.

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and ß1 subunits of Na+ /K+ -ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem ). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem . Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of ß-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.

A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation.

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and ß-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity.

Crim1 is required for maintenance of the ocular lens epithelium.

The development and growth of the vertebrate ocular lens is dependent on the regulated proliferation of an anterior monolayer of epithelial cells, and their subsequent differentiation into elongate fiber cells. The growth factor rich ocular media that bathes the lens mediates these cellular processes, and their respective intracellular signaling pathways are in turn regulated to ensure that the proper lens architecture is maintained. Recent studies have proposed that Cysteine Rich Motor Neuron 1 (Crim1), a transmembrane protein involved in organogenesis of many tissues, might influence cell adhesion, polarity and proliferation in the lens by regulating integrin-signaling. Here, we characterise the lens and eyes of the Crim1KST264 mutant mice, and show that the loss of Crim1 function in the ocular tissues results in inappropriate differentiation of the lens epithelium into fiber cells. Furthermore, restoration of Crim1 levels in just the lens tissue of Crim1KST264 mice is sufficient to ameliorate most of the dysgenesis observed in the mutant animals. Based on our findings, we propose that tight regulation of Crim1 activity is required for maintenance of the lens epithelium, and its depletion leads to ectopic differentiation into fiber cells, dramatically altering lens structure and ultimately leading to microphthalmia and aphakia.

The ßγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability.

ßγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca2+-binding proteins with a large diversity and variable properties in Ca2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal ßγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal ßγ-crystallin domain was deleted (LS-PIPLCΔCRY) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLCΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

A Transition Metal-Binding, Trimeric ßγ-Crystallin from Methane-Producing Thermophilic Archaea, Methanosaeta thermophila.

ßγ-Crystallins are important constituents of the vertebrate eye lens, whereas in microbes, they are prevalent as Ca2+-binding proteins. In archaea, ßγ-crystallins are conspicuously confined to two methanogens, viz., Methanosaeta and Methanosarcina. One of these, i.e., M-crystallin from Methanosarcina acetivorans, has been shown to be a typical Ca2+-binding ßγ-crystallin. Here, with the aid of a high-resolution crystal structure and isothermal titration calorimetry, we report that "Methallin", a ßγ-crystallin from Methanosaeta thermophila, is a trimeric, transition metal-binding protein. It binds Fe, Ni, Co, or Zn ion with nanomolar affinity, which is consistent even at 55 °C, the optimal temperature for the methanogen's growth. At the center of the protein trimer, the metal ion is coordinated by six histidines, two from each protomer, leading to an octahedral geometry. Small-angle X-ray scattering analysis confirms that the trimer seen in the crystal lattice is a biological assembly; this assembly dissociates to monomers upon removal of the metal ion. The introduction of two histidines (S17H/S19H) into a homologous ßγ-crystallin, Clostrillin, allows it to bind nickel at the introduced site, though with micromolar affinity. However, because of the lack of a compatible interface, nickel binding could not induce trimerization, affirming that Methallin is a naturally occurring trimer for high-affinity transition metal binding. While ßγ-crystallins are known to bind Ca2+ and form homodimers and oligomers, the transition metal-binding, trimeric Methallin is a new paradigm for ßγ-crystallins. The distinct features of Methallin, such as nickel or iron binding, are also possible imprints of biogeochemical changes during the period of its origin.

Differences in solution dynamics between lens ß-crystallin homodimers and heterodimers probed by hydrogen-deuterium exchange and deamidation.

BACKGROUND: Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. ßB2 crystallin in the lens of the eye readily forms dimers with other ß-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. METHODS: Structures of ßA3 and ßB2 crystallin homodimers and the ßA3/ßB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen-deuterium exchange with mass spectrometry. We further mimicked deamidation in ßB2 and probed the effect on the ßA3/ßB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. RESULTS: Both ßA3 and ßB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of ßB2 was supported by the identification of chemical crosslinks between lysines in ßB2 within the heterodimer that were inconsistent with ßB2's extended homodimeric structure. The compact structure of ßA3 was supported by an overall decrease in mobility of ßA3 in the heterodimer detected by NMR. In ßB2, peptides 70-84 and 121-134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in ßA3, 97-109 and 134-149, had 25-50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between ßB2 and ßA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in ßB2 at this predicted interface led to a less compact ßB2 in the heterodimer. The more compact structure of the ßA3/ßB2 heterodimer was also more heat stable than either of the homodimers. CONCLUSIONS: The major structural proteins in the lens, the ß-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of ßB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. GENERAL SIGNIFICANCE: Understanding complex hetero-oligomer interactions between ß-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Ca2+ and ßγ-crystallins: An affair that did not last?

BACKGROUND: During the last three decades, lens ß- and γ-crystallins have found a huge number of kin from numerous taxonomical sources. Most of these proteins from invertebrates and microbes have been demonstrated or predicted to bind Ca2+ involving a distinct double-clamp motif, which is largely degenerated in lens homologues. SCOPE OF REVIEW: The various aspects of transformation of ßγ-crystallins from a quintessential Ca2+-binding protein into a primarily structural molecule have been reviewed. MAJOR CONCLUSIONS: In lens members of ßγ-crystallins, the residues involved in Ca2+ binding have diverged considerably from the classical consensus with consequent reduction in their Ca2+-binding properties. This evolutionary change is congenial to their new role as robust constituents of lens. The exact functions of the residual affinity for Ca2+ are yet to be established. GENERAL SIGNIFICANCE: This review highlights the significance of reduction in Ca2+-binding ability of the ßγ-crystallins for lens physiology and why this residual affinity may be functionally important. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Preparation and Characterization of Biomimetic ß-Lens Crystallins Using Single-Chain Polymeric Nanoparticles.

Presbyopia, the inability to focus at arm's length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (∼40%) of globular proteins referred to as α, ß, and γ crystallins, of which ß is the most abundant. In this study, we focus on the synthesis and characterization of a ß-crystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analogue. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and cross-linked with diamines to form nanoparticles. The composition and cross-linking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of ßhigh lens crystallins. The refractive indices and densities of the nanoparticles' dispersion at different concentrations matched that of ßhigh lens crystallins, and the viscosity of the nanoparticles approached that of ßhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial (ppRPE) cells and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacements for the crystallins in developing an accommodating intraocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.

Evidence of Highly Conserved ß-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens.

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in ß-crystallins (except ßA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H(2)O(2), whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the ß-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.