Structural and functional analysis of SNP rs76740365 G>A in exon-3 of the alpha A-crystallin gene in lens epithelial cells.

Purpose: To clarify the effect of a previously identified single nucleotide polymorphism (SNP; rs76740365 G>A) in the exon-3 of the alpha A-crystallin (CRYAA) gene on the properties of CRYAA and to investigate its function in human lens epithelial cells (HLECs). Methods: The human recombinant wild-type and mutant CRYAA (E156K) were constructed, and the molecular weight was measured by mass spectrometry. The structural changes induced by E156K mutation were analyzed by UV circular dichroism spectra and intrinsic tryptophan fluorescence and were predicted using Schrödinger software. The chaperone-like ability of wild-type and E156K mutant CRYAA was invested against the heat-induced aggregation of ßL-crystallin and the DTT-induced aggregation of insulin. HLECs expressing wild-type and mutated CRYAA were subjected to quantitative PCR (qPCR) and western blot. Cell apoptosis was determined using flow cytometry analysis, and the expression of apoptosis-related proteins were determined using western blot. Results: The mass spectrometric detection revealed that E156K mutation had no significant effect on the apparent molecular mass of the CRYAA oligomeric complex. Evaluation of the structures of the CRYAA indicated that E156K mutation did not significantly affect the secondary structures, while causing perturbations of the tertiary structure. The mutant CRYAA displayed an increase in chaperone-like activity, which might be related to the increase of the surface hydrophobicity. We also predicted that E156K mutation would induce a change from negatively charged surface to positively charged, which was the possible reason for the disturbance to the surface hydrophobicity. Transfection studies of HLECs revealed that the E156K mutant induced anti-apoptotic function in HLECs, which was possibly associated with the activation of the p-AKT signal pathway and downregulation of Casepase3. Conclusions: Taken together, our results for the first time showed that E156K mutation in CRYAA associated with ARC resulted in enhanced chaperone-like function by inducing its surface hydrophobicity, which was directly related to the activation of its anti-apoptotic function.

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds.

The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound), and their simultaneous application on the structural and functional features of human αA-crystallin (αA-Cry) using various spectroscopy techniques. Additionally, the surface tension and oligomer size distribution of the treated and untreated protein were tested using tensiometric analysis and dynamic light scattering, respectively. Our results indicated that the reaction of PON and MGO with human αA-Cry leads to the formation of new chromophores, alterations in the secondary to quaternary protein structure, reduction in the size of protein oligomers, and significant enhancement in the chaperone activity of αA-Cry. To reverse the effects of the tested compounds, ascorbic acid and glutathione (main components of lens antioxidant defense system) were applied. As expected, the two antioxidant compounds significantly prevented formation of high molecular weight aggregates of αA-Cry (according to SDS-PAGE). Our results suggest that the lens antioxidant defense system, in particular, glutathione, may provide a strong protection against rapid incidence and progression of diabetic cataract by preventing the destructive reactions of highly reactive DM-associated metabolites.

The Aggregation of αB-Crystallin under Crowding Conditions Is Prevented by αA-Crystallin: Implications for α-Crystallin Stability and Lens Transparency.

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.

Influence of the conformations of αA-crystallin peptides on the isomerization rates of aspartic acid residues.

The isomerization rate of aspartic acid (Asp) residue is known to be affected by the three-dimensional structures of peptides and proteins. Although the isomerized Asp residues were experimentally observed, structural features which affect the isomerization cannot be elucidated sufficiently because of protein denaturation and aggregation. In this study, molecular dynamics (MD) simulations were conducted on three αA-crystallin peptides (T6, T10, and T18), each containing a single Asp residue with different isomerization rate (T18 > T6 > T10) to clarify the structural factors of Asp isomerization tendency. For MD trajectories, distances between side-chain carboxyl carbon of Asp and main-chain amide nitrogen of (n + 1) residue (Cγ-N distances), root mean square fluctuations (RMSFs), and polar surface areas for main-chain amide nitrogen of (n + 1) residues (PSAN) were calculated, because these structural features are considered to relate to the formations of cyclic imide intermediates. RMSFs and PSAN are indexes of peptide backbone flexibilities and solvent exposure of the amide nitrogen, respectively. The average Cγ-N distances of T10 was longer than those of the other two peptides. In addition, the peptide containing Asp residue with a higher isomerization rate showed higher flexibility of the peptide backbone around the Asp residue. PSAN for amide nitrogen in T18 were much larger than those of other two peptides. The computational results suggest that Asp-residue isomerization rates are affected by these factors.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

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.

A single Asp isomer substitution in an αA-crystallin-derived peptide induces a large change in peptide properties.

The eye lens is mainly composed of crystallins, which undergo modifications such as oxidation, deamidation and isomerization with aging. Asp58, Asp76, Asp84, and Asp151 residues of αA-crystallin are site-specifically isomerized to L-iso, D-, and D-iso isomers in aged-related cataract lenses. In addition, an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human αA-crystallin, is detected in aged lens. This peptide induces protein aggregation and causes loss of the chaperone function of α-crystallin. The αA66-80 peptide contains Asp76, but it is not known whether isomerization of Asp76 in αA66-80 specifically induces protein aggregation or affects α-crystallin function. Using Fmoc-based solid-phase synthesis, here we synthesized four αA66-80 peptides, each containing L-, L-iso, D-, or D-isoAsp at position 76, and compared their structures and properties. Normal αA66-80 peptide containing the L-Asp76 isomer increased the EDTA-induced aggregation of ADH protein, DTT-induced aggregation of insulin, and heat-induced aggregation of ßL-crystallin. αA66-80 peptide containing D- or D-isoAsp76 had similar or no effects on the aggregation of these proteins. By contrast, αA66-80 peptide containing L-isoAsp76 inhibited the aggregation of all three proteins, indicating that it has chaperone activity. With regard to secondary structure, αA66-80 peptide containing the L-, D-, or D-isoAsp76 isomer had random-coil structure, whereas αA66-80 peptide containing L-isoAsp76 had ß-sheet like structure. A Thioflavin T (ThT) assay indicated that only the L-isoAsp-containing αA66-80 peptide has ß-sheet structure and generates amyloid fibrils. Collectively, these observations indicate that isomerization of Aps76 to the Lß isomer endows ß-sheet structure and chaperone function on this peptide.

Hydrophobic residues of melittin mediate its binding to αA-crystallin.

The molecular chaperone αA-crystallin, mainly localized in the human ocular lens, is believed to protect the lens from opacification and cataract, by suppressing the aggregation of the other lens proteins. The present study provides structural and thermodynamic insights into the ability of human αA-crystallin (HAA) to bind to its partially unfolded clients in the lens, using a small peptide, melittin from bee venom, as a model client. We characterized the thermodynamic parameters of the binding process between melittin and HAA through isothermal titration calorimetry (ITC), and found the binding to be endothermic and entropy-driven. We identified the amino acids in melittin important for binding to HAA by saturation-transfer difference (STD) nuclear magnetic resonance (NMR) experiments, and analysis of NMR line broadening upon titration of melittin with HAA. Our results suggest that hydrophobic residues Ile17 and Ile20 on the C-terminal region of melittin are in close contact with HAA in the melittin-HAA complex. Information obtained from NMR experiments was used to generate structural models of the melittin-HAA complex by molecular docking with high-ambiguity driven docking (HADDOCK). Structural models of the melittin-HAA complex reveal important principles underlying the interaction of HAA with its clients.

The structure and oxidation of the eye lens chaperone αA-crystallin.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.

The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins.

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5-7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.

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

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

Structural and functional consequences of age-related isomerization in α-crystallins.

Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here, we examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity.

Localization of αA-Crystallin in Rat Retinal Müller Glial Cells and Photoreceptors.

Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as "living light guides" as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.

Structural and functional alteration of human αA-crystallin after exposure to full spectrum solar radiation and preventive role of lens antioxidants.

The chronically exposure of eye lenses to ultra violet and visible light of the solar radiation is an important risk factor for development of the senile cataract diseases. Various photosensitizer molecules including riboflavin (RF) play a significant role in photo-oxidative damages of lens proteins underlying development of opacity in the lenticular tissues. In the current study, RF-mediated photo-oxidation of human αA-crystallin (αA-Cry) was assessed using SDS-PAGE analysis, dynamic light scattering and other spectroscopic assessments. The RF-photosensitized reactions led to non-disulfide covalent cross-linking, oligomerization and significant structural changes in αA-Cry. The photo-damaging of αA-Cry under solar radiation was also accompanied by the reduction in both Trp and Tyr fluorescence intensities which followed by the formation of new photosensitizer chromophores. The solvent exposed hydrophobic patches, secondary structures and chaperone-like activity of αA-Cry were significantly altered after exposure to the solar radiation in the presence of RF. Although glutathione and ascorbate were capable to partially protect the photo-induced structural damages of human αA-Cry, they also disrupted its chaperone function when co-exposed with this protein to the solar radiation. Also, the most promising data were obtained with cysteine which its availability in the lenticular tissues is a rate limiting factor for the biosynthesis of glutathione. Overall our results suggest that glutathione and ascorbate, as the major anti-oxidant compounds within lenticular tissues, demonstrate controversial effect on structure and chaperone-like activity of human αA-Cry. Elucidation of this effect may demand further experiments.

αA-crystallin-derived minichaperone stabilizes αAG98R-crystallin by affecting its zeta potential.

Purpose: The G98R mutant of αA-crystallin is associated with the development of presenile cataracts. In vitro, the recombinant mutant protein exhibits altered structural and functional characteristics, along with the propensity to aggregate by itself and precipitate. Previously, we have reported that the N-terminal aspartate substituted form of the antiaggregation peptide, D71FVIFLDVKHFSPEDLTVK88 (αA-minichaperone or mini-αA) prevented aggregation of αAG98R. However, the mechanism of stabilization of αAG98R from aggregation is not fully understood. The purpose of this study was to determine whether the surface charge (zeta (ζ) potential) of αAG98R in the presence of the peptide chaperone contributed to the stabilization of mutant protein, and to identify the sites of interaction between αAG98R and the peptide chaperone. Methods: Wild-type αA-crystallin (αAWT) and recombinant mutant αAG98R were purified from Escherichia coli BL21(DE3)pLysS cells. The ζ potential values of αA-crystallins in the presence or absence of αA-minichaperone and purified protein-peptide complexes were estimated in a ζ potential analyzer. Potential regions within αAG98R that bind the αA-minichaperone were investigated by incubating the protein with a photoactivable minichaperone variant, followed by mass spectrometric analysis. Results: Binding of the αA-minichaperone to aggregation-prone αAG98R was accompanied by an increase in the ζ potential from -15.19±0.870 mV corresponding to αAG98R alone to -28.64±1.640 mV for the purified complex. Mass spectrometric analysis identified 1MDVTIQHPWFK11, 13TLGPFYPSR21, 55TVLDSGISEVR65, and 113EFHRR117 as the αA-minichaperone-binding regions in αAG98R. The results suggest the involvement of the N-terminal region and the α-crystallin domain in the peptide-mediated stabilization of αAG98R. Conclusions: The αA-crystallin-derived minichaperone stabilizes αAG98R by compensating its lost surface charge. Methods for increasing the ζ potential of aggregating proteins can be a potential approach for therapy to protein aggregation diseases.

Characterization of an N-terminal mutant of αA-crystallin αA-R21Q associated with congenital cataract.

Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a "mutational hotspot," with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin - αA-R21L and αA-R21W have been associated with congenital cataract. A third mutant of R21, αA-R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA-R21Q mutation on αA-crystallin function, recombinant αA-R21Q was expressed, purified and characterized in vitro. Compared to wild-type αA-crystallin, the recombinant αA-R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA-R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA-R21Q homo-oligomers and hetero-oligomers also prevented H2O2-induced apoptosis in ARPE-19 cells. Taken together, αA-R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.

Importance of the positively charged residue at position 54 to the chaperoning function, conformational stability and amyloidogenic nature of human αA-crystallin.

Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry.

Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils.

Amyloid fibril formation occurs from a wide range of peptides and proteins and is typically associated with a loss of protein function and/or a gain of toxic function, as the native structure of the protein undergoes major alteration to form a cross ß-sheet array. It is now well recognised that some amyloid fibrils have a biological function, which has led to increased interest in the potential that these so-called functional amyloids may either retain the function of the native protein, or gain function upon adopting a fibrillar structure. Herein, we investigate the molecular chaperone ability of α-crystallin, the predominant eye lens protein which is composed of two related subunits αA- and αB-crystallin, and its capacity to retain and even enhance its chaperone activity after forming aggregate structures under conditions of thermal and chemical stress. We demonstrate that both eye lens α-crystallin and αB-crystallin (which is also found extensively outside the lens) retain, to a significant degree, their molecular chaperone activity under conditions of structural change, including after formation into amyloid fibrils and amorphous aggregates. The results can be related directly to the effects of aging on the structure and chaperone function of α-crystallin in the eye lens, particularly its ability to prevent crystallin protein aggregation and hence lens opacification associated with cataract formation.

RNA aptamers targeted for human αA-crystallin do not bind αB-crystallin, and spare the α-crystallin domain.

The molecular chaperones, α-crystallins, belong to the small heat shock protein (sHSP) family and prevent the aggregation and insolubilization of client proteins. Studies in vivo have shown that the chaperone activity of the α-crystallins is raised or lowered in various disease states. Therefore, the development of tools to control chaperone activity may provide avenues for therapeutic intervention, as well as enable a molecular understanding of chaperone function. The major human lens α-crystallins, αA- (HAA) and αB- (HAB), share 57% sequence identity and show similar activity towards some clients, but differing activities towards others. Notably, both crystallins contain the "α-crystallin domain" (ACD, the primary client binding site), like all other members of the sHSP family. Here we show that RNA aptamers selected for HAA, in vitro, exhibit specific affinity to HAA but do not bind HAB. Significantly, these aptamers also exclude the ACD. This study thus demonstrates that RNA aptamers against sHSPs can be designed that show high affinity and specificity - yet exclude the primary client binding region - thereby facilitating the development of RNA aptamer-based therapeutic intervention strategies.

α-Crystallins Are Small Heat Shock Proteins: Functional and Structural Properties.

During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αA- and αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein-protein interactions.