Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry.

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Butein Inhibits the Glycation of α-Crystallin: An Approach in Prevention of Retinopathy.

The aggregation of lens proteins induced by glycation is one of the key drivers of diabetic retinopathy and development of diabetic cataracts. Moreover, glycation also causes numerous alterations not only to the tertiary structure of lens proteins but also to serum proteins. There are also evidences of covalent crosslinking among lens crystallins resulting in development of cataract. In this article, the inhibitory potential of butein was tested against the glucose induced glycation and the aggregation α-crystallin (α-cry). The results showed that there was inhibition of advanced glycation products (78.28%) and early glycation products (86.30%) following the treatment of butein. Additionally, the presence of butein caused a significant improvement in the tested biochemical markers of glycation. The treatment with butein reduced the free lysine modification to 23.67%. The secondary and tertiary structural distortions of α-cry were also protected. The mechanism of inhibition further investigated at the molecular level using biophysical and computational techniques. The interaction data showed the butein exhibited strong affinity towards the α-cry. The binding event was entropically driven and energetically favourable. The Gibb's free energy of the interaction was found to be -5.99 to -7.17 kcal mol-1. The binding site of butein in α-cry was deciphered by molecular docking and the dynamics was studied using molecular dynamics (MD) simulations. The simulation data showed that butein formed stable complex with α-cry under physiological conditions. Most of the tested parameters from molecular simulations, such as secondary structure, was found to be stable. The data clearly show the potential of butein in inhibiting the glycation induced aggregation of α-cry and hence can be developed as useful inhibitor in the management of diabetic cataract and retinopathy.

α-Crystallins in the Vertebrate Eye Lens: Complex Oligomers and Molecular Chaperones.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged ß- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.

Protein Aggregation and Cataract: Role of Age-Related Modifications and Mutations in α-Crystallins.

* The article is published as a part of the Special Issue "Protein Misfolding and Aggregation in Cataract Disorders" (Vol. 87, No. 2). ** To whom correspondence should be addressed. Cataract is a major cause of blindness. Due to the lack of protein turnover, lens proteins accumulate age-related and environmental modifications that alter their native conformation, leading to the formation of aggregation-prone intermediates, as well as insoluble and light-scattering aggregates, thus compromising lens transparency. The lens protein, α-crystallin, is a molecular chaperone that prevents protein aggregation, thereby maintaining lens transparency. However, mutations or post-translational modifications, such as oxidation, deamidation, truncation and crosslinking, can render α-crystallins ineffective and lead to the disease exacerbation. Here, we describe such mutations and alterations, as well as their consequences. Age-related modifications in α-crystallins affect their structure, oligomerization, and chaperone function. Mutations in α-crystallins can lead to the aggregation/intracellular inclusions attributable to the perturbation of structure and oligomeric assembly and resulting in the rearrangement of aggregation-prone regions. Such rearrangements can lead to the exposure of hitherto buried aggregation-prone regions, thereby populating aggregation-prone state(s) and facilitating amorphous/amyloid aggregation and/or inappropriate interactions with cellular components. Investigations of the mutation-induced changes in the structure, oligomer assembly, aggregation mechanisms, and interactomes of α-crystallins will be useful in fighting protein aggregation-related diseases.

Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Biochemistry of Eye Lens in the Norm and in Cataractogenesis.

The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.

Isomerization as the secret Achilles' heel of long-lived proteins.

Crystallin proteins, the dominant constituents of the eye lens, are prototypes of long-lived proteins. Such proteins can accumulate harmful modifications over their life span that render them prone to aggregation, which, in the case of lens crystallin, contributes to cataract formation. Lyon et al. now explore the structural and functional consequences of amino acid isomerization in α-crystallins using mass spectrometry, molecular dynamics simulations, and other strategies. Their results highlight the potential deleterious effects of these under-detected modifications on protein structural integrity and function.

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked ß- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-ß-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

A meta-analysis and review examining a possible role for oxidative stress and singlet oxygen in diverse diseases.

From kinetic data (k, T) we calculated the thermodynamic parameters for various processes (nucleation, elongation, fibrillization, etc.) of proteinaceous diseases that are related to the ß-amyloid protein (Alzheimer's), to tau protein (Alzheimer's, Pick's), to α-synuclein (Parkinson's), prion, amylin (type II diabetes), and to α-crystallin (cataract). Our calculations led to ΔG≠ values that vary in the range 92.8-127 kJ mol-1 at 310 K. A value of ∼10-30 kJ mol-1 is the activation energy for the diffusion of reactants, depending on the reaction and the medium. The energy needed for the excitation of O2 from the ground to the first excited state (1Δg, singlet oxygen) is equal to 92 kJ mol-1 So, the ΔG≠ is equal to the energy needed for the excitation of ground state oxygen to the singlet oxygen (1Δg first excited) state. The similarity of the ΔG≠ values is an indication that a common mechanism in the above disorders may be taking place. We attribute this common mechanism to the (same) role of the oxidative stress and specifically of singlet oxygen, (1Δg), to the above-mentioned processes: excitation of ground state oxygen to the singlet oxygen, 1Δg, state (92 kJ mol-1), and reaction of the empty π* orbital with high electron density regions of biomolecules (∼10-30 kJ mol-1 for their diffusion). The ΔG≠ for cases of heat-induced cell killing (cancer) lie also in the above range at 310 K. The present paper is a review and meta-analysis of literature data referring to neurodegenerative and other disorders.

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.

Therapeutic potential of α-crystallin.

BACKGROUND: The findings that α-crystallins are multi-functional proteins with diverse biological functions have generated considerable interest in understanding their role in health and disease. Recent studies have shown that chaperone peptides of α-crystallin could be delivered into cultured cells and in experimental animals with beneficial effects against protein aggregation, oxidation, inflammation and apoptosis. SCOPE OF REVIEW: In this review, we will summarize the latest developments on the therapeutic potential of α-crystallins and their functional peptides. MAJOR CONCLUSIONS: α-Crystallins and their functional peptides have shown significant favorable effects against several diseases. Their targeted delivery to tissues would be of great therapeutic benefit. However, α-crystallins can also function as disease-causing proteins. These seemingly contradictory functions must be carefully considered prior to their therapeutic use. GENERAL SIGNIFICANCE: αA and αB-Crystallin are members of the small heat shock protein family. These proteins exhibit molecular chaperone and anti-apoptotic activities. The core crystallin domain within these proteins is largely responsible for these prosperities. Recent studies have identified peptides within the crystallin domain of both α- and αB-crystallins with remarkable chaperone and anti-apoptotic activities. Administration of α-crystallin or their functional peptides has shown substantial inhibition of pathologies in several diseases. However, α-crystallins have been shown to promote disease-causing pathways. These two sides of the proteins are discussed in this review. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Interaction of α-crystallin with some small molecules and its effect on its structure and function.

BACKGROUND: α-Crystallin acts like a molecular chaperone by interacting with its substrate proteins and thus prevents their aggregation. It also interacts with various kinds of small molecules that affect its structure and function. SCOPE OF REVIEW: In this article we will present a review of work done with respect to the interaction of ATP, peptide generated from lens crystallin and other proteins and some bivalent metal ions with α-crystallin and discuss the role of these interactions on its structure and function and cataract formation. We will also discuss the interaction of some hydrophobic fluorescence probes and surface active agents with α-crystallin. MAJOR CONCLUSIONS: Small molecule interaction controls the structure and function of α-crystallin. ATP and Zn+2 stabilize its structure and enhance chaperone function. Therefore the depletion of these small molecules can be detrimental to maintenance of lens transparency. However, the accumulation of small peptides due to protease activity in the lens can also be harmful as the interaction of these peptides with α-crystallin and other crystallin proteins in the lens promotes aggregation and loss of lens transparency. The use of hydrophobic probe has led to a wealth of information regarding the location of substrate binding site and nature of chaperone-substrate interaction. Interaction of surface active agents with α-crystallin has helped us to understand the structural stability and oligomeric dissociation in α-crystallin. GENERAL SIGNIFICANCE: These interactions are very helpful in understanding the mechanistic details of the structural changes and chaperone function of α-crystallin. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Alpha-crystallin-derived peptides as therapeutic chaperones.

BACKGROUND: The demonstration of chaperone-like activity in peptides (mini-chaperones) derived from α-crystallin's chaperone region has generated significant interest in exploring the therapeutic potential of peptide chaperones in diseases of protein aggregation. Recent studies in experimental animals show that mini-chaperones could reach intended targets and alter the disease phenotype. Although mini-chaperones show potential benefits against protein aggregation diseases, they do tend to form aggregates on storage. There is thus a need to fine-tune peptide chaperones to increase their solubility, pharmacokinetics, and biological efficacy. SCOPE OF REVIEW: This review summarizes the properties and the potential therapeutic roles of mini-chaperones in protein aggregation diseases and highlights some of the refinements needed to increase the stability and biological efficacy of mini-chaperones while maintaining or enhancing their chaperone-like activity against precipitation of unfolding proteins. MAJOR CONCLUSIONS: Mini-chaperones suppress the aggregation of proteins, block amyloid fibril formation, stabilize mutant proteins, sequester metal ions, and exhibit antiapoptotic properties. Much work must be done to fine-tune mini-chaperones and increase their stability and biological efficacy. Peptide chaperones could have a great therapeutic value in diseases associated with protein aggregation and apoptosis. GENERAL SIGNIFICANCE: Accumulation of misfolded proteins is a primary cause for many age-related diseases, including cataract, macular degeneration, and various neurological diseases. Stabilization of native proteins is a logical therapeutic approach for such diseases. Mini-chaperones, with their inherent antiaggregation and antiapoptotic properties, may represent an effective therapeutic molecule to prevent the cascade of protein conformational disorders. Future studies will further uncover the therapeutic potential of mini-chaperones. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Structure and function of α-crystallins: Traversing from in vitro to in vivo.

BACKGROUND: The two α-crystallins (αA- and αB-crystallin) are major components of our eye lenses. Their key function there is to preserve lens transparency which is a challenging task as the protein turnover in the lens is low necessitating the stability and longevity of the constituent proteins. α-Crystallins are members of the small heat shock protein family. αB-crystallin is also expressed in other cell types. SCOPE OF THE REVIEW: The review summarizes the current concepts on the polydisperse structure of the α-crystallin oligomer and its chaperone function with a focus on the inherent complexity and highlighting gaps between in vitro and in vivo studies. MAJOR CONCLUSIONS: Both α-crystallins protect proteins from irreversible aggregation in a promiscuous manner. In maintaining eye lens transparency, they reduce the formation of light scattering particles and balance the interactions between lens crystallins. Important for these functions is their structural dynamics and heterogeneity as well as the regulation of these processes which we are beginning to understand. However, currently, it still remains elusive to which extent the in vitro observed properties of α-crystallins reflect the highly crowded situation in the lens. GENERAL SIGNIFICANCE: Since α-crystallins play an important role in preventing cataract in the eye lens and in the development of diverse diseases, understanding their mechanism and substrate spectra is of importance. To bridge the gap between the concepts established in vitro and the in vivo function of α-crystallins, the joining of forces between different scientific disciplines and the combination of diverse techniques in hybrid approaches are necessary. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Differential role of arginine mutations on the structure and functions of α-crystallin.

BACKGROUND: α-Crystallin is a major protein of the eye lens in vertebrates. It is composed of two subunits, αA- and αB-crystallin. α-Crystallin is an oligomeric protein having these two subunits in 3:1 ratio. It belongs to small heat shock protein family and exhibits molecular chaperone function, which plays an important role in maintaining the lens transparency. Apart from chaperone function, both subunits also exhibit anti-apoptotic property. Comparison of their primary sequences reveals that αA- and αB-crystallin posses 13 and 14 arginine residues, respectively. Several of them undergo mutations which eventually lead to various eye diseases such as congenital cataract, juvenile cataract, and retinal degeneration. Interestingly, many arginine residues of these subunits are modified during glycation and even some are truncated during aging. All these facts indicate the importance of arginine residues in α-crystallin. SCOPE OF REVIEW: In this review, we will emphasize the recent in vitro and in vivo findings related to congenital cataract causing arginine mutations in α-crystallin. MAJOR CONCLUSIONS: Congenital cataract causing arginine mutations alters the structure and decreases the chaperone function of α-crystallin. These mutations also affect the lens morphology and phenotypes. Interestingly, non-natural arginine mutations (generated for mimicking the glycation and truncation environment) improve the chaperone function of α-crystallin which may play an important role in maintaining the eye lens transparency during aging. GENERAL SIGNIFICANCE: The neutralization of positive charge on the guanidino group of arginine residues is not always detrimental to the functionality of α-crystallin. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Alpha crystallins in the retinal pigment epithelium and implications for the pathogenesis and treatment of age-related macular degeneration.

BACKGROUND: αA- and αB crystallins are principal members of the small heat shock protein family and elicit both a cell protective function and a chaperone function. α-Crystallins have been found to be prominent proteins in normal and pathological retina emphasizing the importance for in-depth understanding of their function and significance. SCOPE OF REVIEW: Retinal pigment epithelial cells (RPE) play a vital role in the pathogenesis of age-related macular degeneration (AMD). This review addresses a number of cellular functions mediated by α-crystallins in the retina. Prominent expression of αB crystallin in mitochondria may serve to protect cells from oxidative injury. αB crystallin as secretory protein via exosomes can offer neuroprotection to adjacent RPE cells and photoreceptors. The availability of chaperone-containing minipeptides of αB crystallin could prove to be a valuable new tool for therapeutic treatment of retinal disorders. MAJOR CONCLUSIONS: α-Crystallins are expressed in cytosol and mitochondria of RPE cells and are regulated during oxygen-induced retinopathy and during development. α-Crystallins protect RPE from oxidative-and ER stress-induced injury and autophagy. αB-Crystallin is a modulator of angiogenesis and vascular endothelial growth factor. αB Crystallin is secreted via exosomal pathway. Minichaperone peptides derived from αB Crystallin prevent oxidant induced cell death and have therapeutic potential. GENERAL SIGNIFICANCE: Overall, this review summarizes several novel properties of α-crystallins and their relevance to maintaining normal retinal function. In particular, the use of α-crystallin derived peptides is a promising therapeutic strategy to combat retinal diseases such as AMD. This article is part of a Special Issue entitled Crystallin biochemistry in health and disease.

Protective role of antioxidant compounds against peroxynitrite-mediated modification of R54C mutant αA-crystallin.

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has endogenous production in the eye ball and contributes to a variety of ocular disorders. In the current study the structural characteristics, chaperone-like activity and conformational stability of R54C mutant αA-crystallin (αA-Cry) were studied upon modification with PON and in the presence of three antioxidant compounds such as ascorbic acid (ASA), glutathione (GSH) and N-acetylcysteine (NAC) using gel electrophoresis and different spectroscopy methods. The results of both fluorescence analysis and gel electrophoresis suggested that PON modification leads to dityrosine-mediated intermolecular cross-linking of this cataractogenic mutant protein. Also, the propensity of R54C mutant αA-Cry for disulfide cross-linking was increased upon PON modification. In addition, the PON-modified protein indicated structural alteration, reduced chemical stability and different pattern of proteolysis. Upon modification with PON, mutant αA-Cry displayed a significant increase in the chaperone-like activity against aggregation of γ-crystallin and insulin. In addition, different antioxidant compounds indicated a prominent role in neutralizing the PON damaging effects on structural integrity and stability of this protein. The results of this study may highlight the importance of antioxidant-rich foods or potent antioxidant supplements in protection of lens crystallins against PON-mediated structural damages and cataract development.

Hard sphere-like glass transition in eye lens α-crystallin solutions.

We study the equilibrium liquid structure and dynamics of dilute and concentrated bovine eye lens α-crystallin solutions, using small-angle X-ray scattering, static and dynamic light scattering, viscometry, molecular dynamics simulations, and mode-coupling theory. We find that a polydisperse Percus-Yevick hard-sphere liquid-structure model accurately reproduces both static light scattering data and small-angle X-ray scattering liquid structure data from α-crystallin solutions over an extended range of protein concentrations up to 290 mg/mL or 49% vol fraction and up to ca. 330 mg/mL for static light scattering. The measured dynamic light scattering and viscosity properties are also consistent with those of hard-sphere colloids and show power laws characteristic of an approach toward a glass transition at α-crystallin volume fractions near 58%. Dynamic light scattering at a volume fraction beyond the glass transition indicates formation of an arrested state. We further perform event-driven molecular dynamics simulations of polydisperse hard-sphere systems and use mode-coupling theory to compare the measured dynamic power laws with those of hard-sphere models. The static and dynamic data, simulations, and analysis show that aqueous eye lens α-crystallin solutions exhibit a glass transition at high concentrations that is similar to those found in hard-sphere colloidal systems. The α-crystallin glass transition could have implications for the molecular basis of presbyopia and the kinetics of molecular change during cataractogenesis.