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.

Synergistic protection of RGCs by olfactory ensheathing cells and alpha-crystallin through regulation of the Akt/BAD Pathway.

BACKGROUND: Our recent in vivo studies have shown that olfactory ensheathing cells (OECs) and α-crystallin can promote retinal ganglion cell (RGC) survival and axonal regeneration synergistically after optic nerve injury. However, the mechanism is still unknown. OBJECTIVES: Here, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGC survival after H2O2-induced oxidative damage and a crushing injury to the optic nerve in an adult rat model. METHODS: After H2O2-induced oxidative damage, RGC-5 cells were treated with OECs, α-crystallin or a combination of OECs and α-crystallin. Apoptosis of RGC-5 cells was assessed by flow cytometry. Phosphorylated Akt, BAD, and cleaved-caspase3 were detected by Western blot after optic nerve injury in vivo and H2O2-induced RGC-5 oxidative damage in vitro. RESULTS: The results showed that OECs and α-crystallin could both independently inhibit RGC-5 apoptosis (P<0.01), increase the phosphorylation of both Akt and BAD, and decrease the activation of caspase-3 (P<0.01). However, the effect of the combination of both was more significant than either alone. CONCLUSION: These findings indicate that inhibition of superoxide damage to RGCs through regulation of the Akt/BAD pathway is one of the mechanisms by which OECs and α-crystallin promote optic nerve recovery after injury.

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.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.

The effect of Arg on the structure perturbation and chaperone activity of α-crystallin in the presence of the crowding agent, dextran.

α-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses. It has a molecular weight of 20 kDa and is composed of two subunits: αA and αB. α-Crystallin is a member of the small heat shock protein (sHsps) family that has been shown to prevent protein aggregation. Small molecules are organic compounds that have low molecular weight (<800 Da). Arginin (Arg) is a small molecule and has been shown to prevent protein aggregation through interaction with partially folded intermediates. In this study, the effect of Arg on the chaperone activity of α-crystallin in the presence of dextran, as a crowding agent, against ordered and disordered aggregation of different target proteins (α-lactalbumin, ovotransferrin, and catalase) has been investigated. The experiments were done using visible absorption spectroscopy, ThT-binding assay, fluorescence spectroscopy, and CD spectroscopy. The results showed that in amorphous aggregation and amyloid fibril formation, both in the presence and absence of dextran, Arg had a positive effect on the chaperone action of α-crystallin. However, in the presence of dextran, the effect of Arg on the chaperone ability of α-crystallin was less than in its absence. Thus, our result suggests that crowding interior media decreases the positive effect of Arg on the chaperone ability of α-crystallin. This is a very important issue, since we are trying to find a mechanism to protect living cells against the toxic effect of protein aggregation.

Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

AIM: To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. MAIN METHODS: Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. KEY FINDINGS: Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (P<0.05). Moreover, endogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (P<0. 01) and was associated with a decrease in caspase-3 expression (P<0. 05). SIGNIFICANCE: The present study demonstrates that the expression of endogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons.

Novel roles for α-crystallins in retinal function and disease.

α-Crystallins are key members of the superfamily of small heat shock proteins that have been studied in detail in the ocular lens. Recently, novel functions for α-crystallins have been identified in the retina and in the retinal pigmented epithelium (RPE). αB-Crystallin has been localized to multiple compartments and organelles including mitochondria, golgi apparatus, endoplasmic reticulum and nucleus. α-Crystallins are regulated by oxidative and endoplasmic reticulum stress, and inhibit apoptosis-induced cell death. α-Crystallins interact with a large number of proteins that include other crystallins, and apoptotic, cytoskeletal, inflammatory, signaling, angiogenic, and growth factor molecules. Studies with RPE from αB-crystallin deficient mice have shown that αB-crystallin supports retinal and choroidal angiogenesis through its interaction with vascular endothelial growth factor. αB-Crystallin has also been shown to have novel functions in the extracellular space. In RPE, αB-crystallin is released from the apical surface in exosomes where it accumulates in the interphotoreceptor matrix and may function to protect neighboring cells. In other systems administration of exogenous recombinant αB-crystallin has been shown to be anti-inflammatory. Another newly described function of αB-crystallin is its ability to inhibit ß-amyloid fibril formation. α-Crystallin minichaperone peptides have been identified that elicit anti-apoptotic function in addition to being efficient chaperones. Generation of liposomal particles and other modes of nanoencapsulation of these minipeptides could offer great therapeutic advantage in ocular delivery for a wide variety of retinal degenerative, inflammatory and vascular diseases including age-related macular degeneration and diabetic retinopathy.

α-Crystallin promotes rat axonal regeneration through regulation of RhoA/rock/cofilin/MLC signaling pathways.

Intravitreal injection of α-crystallin can promote axons from optic nerve regeneration after crushing in rats. We have previously demonstrated that α-crystallin can counteract the effect of myelin inhibitory factors and stimulate neurite growth. And a common crucial signaling event for myelin inhibitory factors is the activation of RhoA. To investigate whether α-crystallin counteracts the inhibitory effect of myelin inhibitory factors through regulation of RhoA/Rock signaling pathway, α-crystallin (10(-4) g/L) was injected into rat vitreous at the time the optic nerve crushed. The RhoA protein activity and the expression of RhoA and Rock were evaluated after 3 days of optic nerve axotomy. Rock downstream effectors, phosphorylated cofilin, and phosphorylated myosin light chain were detected when retinal neurons were cultured for 3 days. Axonal regeneration and neurites growth of cultured cells were observed also. Our results showed that α-crystallin decreased the RhoA protein activity and the phosphorylation of both cofilin and myosin light chain, and promoted the axonal growth. However, the expression of RhoA and Rock was not affected by α-crystallin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors through the regulation of RhoA/Rock signaling pathway.

Protection against protein aggregation by alpha-crystallin as a mechanism of preconditioning.

Anesthetic preconditioning occurs when cells previously exposed to inhaled anesthetics are protected against subsequent injury. We hypothesize that inhaled anesthetics may cause slight protein misfolding that involves site-specific dehydration, stimulating cytoprotective mechanisms. Human neuroblastoma cells were exposed to ethanol (as the dehydration agent) followed by quantitative analysis of the expression of five heat shock genes: DNAJC5G, CRYAA, HSPB2, HSF4 and HSF2. There was an ethanol-induced upregulation of all genes except HSF4, similar to previous observations using isoflurane. CRYAA (the gene for alphaA-crystallin) exhibited a 23.19 and 17.15-fold increase at 24 and 48 h post ethanol exposure, respectively. Additionally, we exposed glyceraldehyde 3-phosphate dehydrogenase to ethanol, which altered oligomeric subspecies and caused protein aggregation in a concentration-dependent manner. Ethanol-mediated dehydration-induced protein aggregation was prevented by incubation with alpha-crystallin. These data indicate that ethanol mimics the effects of isoflurane presumably through a cellular preconditioning mechanism that involves dehydration-induced protein aggregation.

Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions.

The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research has demonstrated that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. As a result of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.

New focus on alpha-crystallins in retinal neurodegenerative diseases.

The crystallin proteins were initially identified as structural proteins of the ocular lens and have been recently demonstrated to be expressed in normal retina. They are dramatically upregulated by a large range of retinal diseases including diabetic retinopathy, age-related macular degeneration, uveitis, trauma and ischemia. The crystallin family of proteins is composed of alpha-, beta- and gamma-crystallin. Alpha-crystallins, which are small heat shock proteins, have received substantial attention recently. This review summarizes the current knowledge of alpha-crystallins in retinal diseases, their roles in retinal neuron cell survival and retinal inflammation, and the regulation of their expression and activity. Their potential role in the development of new treatments for neurodegenerative diseases is also discussed.

Effect of methylglyoxal modification of human α-crystallin on the structure, stability and chaperone function.

α-Crystallin functions as a molecular chaperone and maintains transparency of eye lens by protecting other lens-proteins. Non-enzymatic glycation of α-crystallin by methylglyoxal, plays a crucial role on its chaperone function and structural stability. Our studies showed that methylglyoxal modification even in lower concentration caused significant decrease in chaperone function of α-crystallin as reflected both in thermal aggregation assay and enzyme refolding assay. Thermal denaturation studies showed drastic reduction of denaturation temperature with increase in the degree of modification. Thermodynamic stability studies by urea denaturation assay reflected a decrease of transition midpoint. Quantitatively we found that ΔG° of native α-crystallin decreased from 21.6 kJ/mol to 10.4 kJ/mol due to 72 h modification by 10 mM methylglyoxal. The surface hydrophobicity of α-crystallin after MG modification, was found to be decreased. Circular dichroism spectroscopy revealed conversion of ß-sheet structure to random coil structure. Significant cross-linking was also observed due to methylglyoxal modification of human α-crystallin.

α- and ß-crystallins modulate the head group order of human lens membranes during aging.

PURPOSE: To examine the physical properties of human lens cell membranes as a function of age. METHODS: The environment of the phospholipid head groups in fiber cell membranes from human lenses, aged 22 to 83 years, was assessed with Laurdan and two-photon confocal microscopy. The effect of mild thermal stress on head group order was studied with lens pairs in which one intact lens was incubated at 50 °C. Dihydrosphingomyelin vesicles were preloaded with Laurdan, α-, ß-, or γ-crystallin was added, and surface fluidity was determined. RESULTS: The membrane head group environment became more fluid with age as indicated by increased water penetration. Furthermore, these changes could be replicated simply by exposing intact human lenses to mild thermal stress; conditions which decreased the concentration of soluble α- and ß-crystallins. Vesicle binding experiments showed that α- and ß-, but not γ-, crystallins markedly affected head group order. CONCLUSIONS: The physical properties of cell membranes in the lens nucleus change substantially with age, and α- and ß-crystallins may modulate this effect. ß-Crystallins may therefore play a role in lens cells, and cells of other tissues, apart from being simple structural proteins. Age-dependent loss of these crystallins may affect membrane integrity and contribute to the dysfunction of lenses in older people.

The role of neutrophils and TNF-related apoptosis-inducing ligand (TRAIL) in bacillus Calmette-Guérin (BCG) immunotherapy for urothelial carcinoma of the bladder.

Intravesical Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunotherapy is a highly effective treatment for carcinoma in situ of the bladder, as well as high-risk nonmuscle invasive urothelial carcinoma of the bladder. Despite over 30 years of clinical experience with BCG, the therapy's mechanism has remained enigmatic. Observations regarding the role of neutrophils in BCG immunotherapy have led to exciting discoveries regarding the potential role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in creating the therapeutic benefit of BCG immunotherapy. In this paper, we will review the scope of the disease, highlight our understanding of the role for BCG in urothelial carcinoma of the bladder, explain the recent discoveries regarding the role of neutrophils and TRAIL in therapy, and theorize on potential future areas of research.

Methionine sulfoxide reductase A (MsrA) restores alpha-crystallin chaperone activity lost upon methionine oxidation.

BACKGROUND: Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and alpha-crystallin which is a two-subunit (alphaA and alphaB) chaperone. Here, we tested whether PMSO formation damages alpha-crystallin chaperone function and whether MsrA could repair PMSO-alpha-crystallin. METHODS: Total alpha-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-alpha-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of alpha-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined. RESULTS: PMSO oxidation of total alpha-crystallin (met 138 of alphaA and met 68 of alphaB) resulted in loss of alpha-crystallin chaperone activity. MsrA treatment of PMSO-alpha-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-alpha-crystallin. CONCLUSIONS: Methionine oxidation damages alpha-crystallin chaperone function and MsrA can repair PMSO-alpha-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of alpha-crystallin methionines in the lens. SIGNIFICANCE: Methionine oxidation of alpha-crystallin in combination with loss of MsrA repair causes loss of alpha-crystallin chaperone function. Since increased PMSO levels and loss of alpha-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.

Phytochelatins inhibit the metal-induced aggregation of alpha-crystallin.

Phytochelatins (PCs) are heavy-metal-binding peptides found in some eukaryotes. This study investigates the use of plant-derived PCs for the inhibition of metal-induced protein aggregation. The results of this study show that PCs inhibit zinc-induced alpha-crystallin aggregation, and suggest that PCs might be useful as anti-cataract agents.

Concurrence of Danish dementia and cataract: insights from the interactions of dementia associated peptides with eye lens alpha-crystallin.

Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens alpha-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease.

Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia.

This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild-type, alphaA-crystallin (-/-), and alphaB-crystallin (-/-) mice received intravitreal injection of 12 nmol (low dose), 33 nmol (intermediate dose) or 60 nmol (high dose) cobalt chloride (CoCl(2)). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24 h, 96 h, and 1 week post-injection, while immunofluorescent stains for alphaA- and alphaB-crystallin were performed 1 week post-injection. The in vitro effects of CoCl(2) on alphaB-crystallin expression in ARPE-19 cells were determined by real time RT-PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of alphaB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl(2) injection in wild-type mice. Retinas of CoCl(2) injected mice showed transient increased expression of HIF-1alpha which was maximal 24h after injection. Intermediate-dose CoCl(2) injection was associated with increased retinal immunofluorescence for both alphaA- and alphaB-crystallin; however, after high-dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl(2) at either intermediate or high dose in alphaA-crystallin (-/-) and alphaB-crystallin (-/-) mice resulted in much more severe retinal degeneration compared to wild-type eyes. A decrease in ARPE-19 total and cytosolic alphaB-crystallin expression with increasing CoCl(2) treatment and an increase in mitochondrial alphaB-crystallin were found. We conclude that lack of alpha-crystallins accentuates retinal degeneration in chemically induced hypoxia in vivo.

Dissection of relationship between small heat shock proteins and mycobacterial diseases.

Mycobacteria belong to a genus which has membership ranging from saprophytes to deadly pathogens that cause several infectious diseases affecting a large population of the world. Among them, tuberculosis and leprosy are the major granulomatous mycobacterial diseases. While there are successes and failures in the fight against these infections, mechanisms of pathogenesis continue to be a challenge to clinicians and biologists alike. Though it is known that both host and bacterial factors are important in the pathogenicity versus protection, all the triggers and responses are not known. Among various bacterial factors, small heat shock proteins (sSHPs) could be important targets for drug development, immunomodulation and serodiagnosis. sSHPs are the molecular chaperones that are believed to act as mantle for the mycobacteria against host's immune attack and facilitate the survival of pathogen in host body. Best studied small heat shock proteins in M. tuberculosis are sSHP16.3 and Acr2 while in M. leprae, it is 18 kD protein antigen. In this review, works on various aspects of small heat shock proteins which fall in 10 to 19 kD range have been summarized and some thoughts about future road-map have been put into.