Impact of α-crystallin protein loss on zebrafish lens development.

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Acceleration of age-induced proteolysis in the guinea pig lens nucleus by in vivo exposure to hyperbaric oxygen: A mass spectrometry analysis.

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66-80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66-80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact ß-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Cataract-causing allele in CRYAA (Y118D) proceeds through endoplasmic reticulum stress in mouse model.

As small heat shock proteins, α-crystallins function as molecular chaperones and inhibit the misfolding and aggregation of ß/γ-crystallins. Genetic mutations of CRYAA are associated with protein aggregation and cataract occurrence. One possible process underlying cataract formation is that endoplasmic reticulum stress (ERS) induces the unfolded protein response (UPR), leading to apoptosis. However, the pathogenic mechanism related to this remains unexplored. Here, we successfully constructed a cataract-causing CRYAA (Y118D) mutant mouse model, in which the lenses of the CRYAA-Y118D mutant mice showed severe posterior rupture, abnormal morphological changes, and aberrant arrangement of crystallin fibers. Histological analysis was consistent with the clinical pathological characteristics. We also explored the pathogenic factors involved in cataract development through transcriptome analysis. In addition, based on key pathway analysis, up-regulated genes in CRYAA-Y118D mutant mice were implicated in the ERS-UPR pathway. This study showed that prolonged activation of the UPR pathway and severe stress response can cause proteotoxic and ERS-induced cell death in CRYAA-Y118D mutant mice.

Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2).

The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid ß (Aß)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aß1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.

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.

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.

Evaluation of the Putative Efficacy of a Methanolic Extract of Ocimum Basilicum in Preventing Disruption of Structural Proteins in an in Vitro System of Selenite-induced Cataractogenesis.

Purpose: To evaluate whether a methanolic extract of Ocimum basilicum (OB) leaves prevented lenticular protein alterations in an in-vitro model of selenite-induced cataractogenesis.Materials and Methods: Transparent lenses extirpated from Wistar rats were divided into three groups: control; selenite only; treated. Control lenses were cultured in Dulbecco's modified Eagle's medium (DMEM) alone, selenite only lenses were cultured in DMEM containing sodium selenite only (100 µM selenite/ml DMEM) and treated lenses were cultured in DMEM containing sodium selenite and the methanolic extract of OB leaves (200 µg of extract/ml DMEM); all lenses were cultured for 24 h and then processed. The parameters assessed in lenticular homogenates were lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) patterns of lenticular proteins, and mRNA transcript and protein levels of αA-crystallin and ßB1-crystallins.Results: Selenite only lenses exhibited alterations in all parameters assessed. Treated lenses exhibited values for these parameters that were comparable to those noted in normal control lenses.Conclusions: The methanolic extract of OB leaves prevented alterations in lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, SDS-PAGE patterns of lenticular proteins, and expression of αA-crystallin and ßB1-crystallin gene and proteins in cultured selenite-challenged lenses. OB may be further evaluated as a promising agent for the prevention of cataract.

Functional Rescue of Cataract-Causing αA-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human αA-Crystallin.

The G98R mutation in αA-crystallin is associated with the onset of presenile cataract and is characterized biochemically by an increased oligomeric mass, altered chaperone function, and loss of structural stability over time. Thus, far, it is not known whether the inherent instability caused by gain-of-charge mutation could be rescued by a compensatory loss of charge mutation elsewhere on the protein. To answer this question, we investigated whether αA-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific "compensatory" mutations in αA-G98R-crystallin, αA-R21Q/G98R, αA-G98R/R116C, and αA-R157Q/G98R. The recombinant proteins were expressed, purified, characterized, and evaluated by circular dichroism (CD), intrinsic fluorescence, and bis-ANS-binding studies. Chaperone-like activities of recombinant proteins were assessed using alcohol dehydrogenase (ADH) and insulin as unfolding substrates. Far-UV CD studies revealed an increased α-helical content in αA-G98R in comparison to αA-WT, αA-R21Q, R157Q, and the double mutants, αA-R21Q/G98R, and αA-R157Q/G98R. Compared to αA-WT, αA-R21Q, and αA-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS-binding) was shown by αA-G98R. Introduction of a second mutation in αA-G98R reduced its bis-ANS-binding activity. Both αA-R21Q/G98R and αA-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than αA-G98R. However, among the three G98R mutants, only αA-R21Q/G98R protected ARPE-19 cells from H2O2-induced cytotoxicity. These results suggest that the lost chaperone-like activity of αA-G98R-crystallin can be rescued by another targeted mutation and that substitution of αA-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved α-crystallin domain of the protein.

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.

Negative charge at aspartate 151 is important for human lens αA-crystallin stability and chaperone function.

Aggregation of lens protein is a major cause of senile cataract. Lens crystallins contain many kinds of modification that accumulate over lifespan. In particular, isomerization of Asp 151 in αA-crystallin has been found in aged lenses; however, its significance is unknown. The purpose of this study was to determine the effects of isomerization of Asp 151 in αA-crystallin. Trypsin digestion followed by liquid chromatography-mass spectrometry analysis of the water-soluble high molecular weight (HMW) fraction from human lens samples showed that isomerization of Asp 151 in αA-crystallin is age-independent, and that 50% of isomerization occurs shortly after birth. However, the extent of Asp 151 isomerization varied with the size of αA-crystallin oligomer species separated from the HMW fraction from aged lens. To evaluate the effects of modification, Asp 151 of αA-crystallin was replaced by glycine, alanine, isoleucine, asparagine, glutamate, or lysine by site-directed mutagenesis. All substitutions except for glutamate decreased heat stability and chaperone function as compared with wild-type αA-crystallin. In particular, abnormal hydrophobicity and alteration of the charge state at Asp 151 caused loss of stability and chaperone activity of αA-crystallin; these properties were recovered to some extent when the mutant protein was mixed 1:1 with wild-type αA-crystallin. The results suggest that, by itself, age-independent isomerization of Asp 151 in αA-crystallin may not contribute to cataract formation. However, the long-term deleterious effect of Asp 151 isomerization on the structure and function of αA-crystallin might cooperatively contribute to the loss of transparency of aged human lens.

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.

RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

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.

A specific phosphorylation regulates the protective role of αA-crystallin in diabetes.

Neurodegeneration is a central aspect of the early stages of diabetic retinopathy, the primary ocular complication associated with diabetes. While progress has been made to improve the vascular perturbations associated with diabetic retinopathy, there are still no treatment options to counteract the neuroretinal degeneration associated with diabetes. Our previous work suggested that the molecular chaperones α-crystallins could be involved in the pathophysiology of diabetic retinopathy; however, the role and regulation of α-crystallins remained unknown. In the present study, we demonstrated the neuroprotective role of αA-crystallin during diabetes and its regulation by its phosphorylation on residue 148. We further characterized the dual role of αA-crystallin in neurons and glia, its essential role for neuronal survival, and its direct dependence on phosphorylation on this residue. These findings support further evaluation of αA-crystallin as a treatment option to promote neuron survival in diabetic retinopathy and neurodegenerative diseases in general.

Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract.

The mammalian eye lens expresses a high concentration of crystallins (α, ß and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.