α-Crystallin, the predominant protein of the eye lens, possesses molecular chaperone activity and antioxidative properties, both of which are essential for maintaining lens transparency. Its chaperone function prevents the formation of light-scattering protein aggregates, while its antioxidative activity mitigates oxidative stress through both direct and indirect mechanisms. However, with aging, α-crystallin undergoes cumulative post-translational modifications and oxidative damage, leading to protein crosslinking and a decline in chaperone efficacy. Notably, α-crystallin exhibits free radical-scavenging activity comparable to that of serum albumin, a well-characterized antioxidant protein. In addition, its ability to bind redox-active metal ions and convert them into redox-inactive forms significantly reduces reactive oxygen species (ROS) generation in vivo. α-Crystallin also interacts with key proteins and signaling pathways involved in oxidative stress responses, further enhancing its multifunctional protective role. This review summarizes current evidence on the antioxidative properties of α-crystallin and their relationship to its chaperone function, highlighting its importance in lens homeostasis and age-related cataract formation.

We previously demonstrated that deletion of the 54FLRAPSW61 sequence, containing the key phosphorylation site serine 59 (S59), resulted in a two-fold reduction in oligomeric mass and a ten-fold enhancement of αB-crystallin's chaperone activity. This study examined whether targeted deletion (ΔS59) or phosphomimetic substitution (S59D) could replicate these effects. Using MALS analysis, we found that the average oligomeric mass decreased from 579 kDa in the wild type (αB-WT) to 556 kDa in αB-ΔS59 and 434 kDa in αB-S59D. Interestingly, the αB-S59A variant had an increased mass of 611 kDa. All variants retained their chaperone function, but their efficiencies varied significantly. Specifically, αB-S59D formed smaller, more polydisperse complexes that effectively suppressed aggregation when interacting with rapidly aggregating substrates. In contrast, αB-ΔS59 and αB-S59A created stable complexes with lysozyme, reducing precipitation and aggregate size. Zeta potential measurements revealed distinct surface charge profiles among the variants; however, no clear correlation was observed between these charges and their chaperone efficiency. Additionally, cytotoxicity assays conducted on ARPE-19 cells under oxidative stress showed that all S59 variants exhibited comparable protective effects against cell death relative to αB-WT. These results indicate that while S59 is not essential for oligomer formation or chaperone function, it plays a crucial role in modulating oligomer size and interactions with various substrates. Notably, the effects of αB-S59D were measurable but did not replicate the enhanced functionality observed with the complete deletion of the 54-61 motif, reinforcing the significance of the N-terminal region.

The G98R mutation in αA-crystallin is associated with presenile cataract development in humans. Previous studies have indicated that mutant proteins altered structure, decreased stability, increased oligomeric size, loss of chaperone-like activity, and susceptibility to proteolysis could be contributing factors to cataract formation. To evaluate the effect of substrate protein interactions with the mutant protein on cataract formation, we have performed chaperone assays with alcohol dehydrogenase (ADH), citrate synthase (CS), and βB2-crystallin (βB2), and analyzed the reaction mixtures by multi-angle light scattering (MALS) analysis. It appears that αAG98R protein initially gets stabilized upon interaction with substrate proteins. Analysis of the chaperone-client protein complexes revealed that wild-type αA-crystallin interacts with substrate proteins to form compact complexes leading to a slight increase in oligomeric mass, whereas αAG98R forms less compact and high molecular weight complexes with the substrate, and the resulting complexes continue to increase in size over time. As a result, the soluble complexes formed initially by the mutant protein begin to scatter light and precipitate. We found that the stability and chaperone activity of the αAG98R can be improved by modifying the protein with low concentrations (50 µM) of methylglyoxal (MGO). Incubation of αAG98R protein (1 mg/ml) under aseptic conditions for 30 days at 37°C resulted in precipitation of the mutant protein. In contrast, mutant protein incubations carried out with 50 µM MGO remained soluble and transparent. SDS-PAGE analysis showed gradual autolysis of the mutant protein in the absence of MGO. The average molar mass of the mutant protein oligomers changed from 7,258 ± 12 kDa to 3,950 ± 08 kDa within 60 min of incubation with MGO. There was no further significant change in the molar mass of mutant protein when tested on day 7 of MGO treatment. Our data suggest that the initial stabilization of αAG98R by substrate proteins could delay congenital cataracts’ appearance, and the uncontrolled long-term interaction amongst mutant subunits and substrate proteins could be the rationale behind presenile cataracts formation. The results also demonstrate the potential benefit of low concentrations of MGO in stabilizing mutant chaperone protein(s).

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ1–42) fibril formation in vitro and suppressed Aβ1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

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 paradigm that cataracts are irreversible and that vision from cataracts can only be restored through surgery has recently been challenged by reports that oxysterols such as lanosterol and 25-hydroxycholesterol can restore vision by binding to αB-crystallin chaperone protein to dissolve or disaggregate lenticular opacities. To confirm this premise, in vitro rat lens studies along with human lens protein solubilization studies were conducted. Cataracts were induced in viable rat lenses cultured for 48 hours in TC-199 bicarbonate media through physical trauma, 10 mM ouabain as Na+/K+ ATPase ion transport inhibitor, or 1 mM of an experimental compound that induces water influx into the lens. Subsequent 48-hour incubation with 15 mM of lanosterol liposomes failed to either reverse these lens opacities or prevent the further progression of cataracts to the nuclear stage. Similarly, 3-day incubation of 47-year old human lenses in media containing 0.20 mM lanosterol or 60-year-old human lenses in 0.25 and 0.50 mM 25-hydroxycholesterol failed to increase the levels of soluble lens proteins or decrease the levels of insoluble lens proteins. These binding studies were followed up with in silico binding studies of lanosterol, 25-hydroxycholesterol, and ATP as a control to two wild type ( 2WJ7 and 2KLR ) and one R120G mutant ( 2Y1Z ) αB-crystallins using standard MOE TM (Molecular Operating Environment) and Schrödinger’s Maestro software. Results confirmed that compared to ATP, both oxysterols failed to reach the acceptable threshold binding scores for good predictive binding to the αB-crystallins. In summary, all three studies failed to provide evidence that lanosterol or 25-hydroxycholesterol have either anti-cataractogenic activity or bind aggregated lens protein to dissolve cataracts.

Purpose: The G98R mutant of alpha A-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, (DFVIFLDVKHFSPEDLTVK88)-F-71 (alpha A-minichaperone or mini-alpha A) prevented aggregation of alpha AG98R. However, the mechanism of stabilization of alpha AG98R from aggregation is not fully understood. The purpose of this study was to determine whether the surface charge (zeta (zeta) potential) of alpha AG98R in the presence of the peptide chaperone contributed to the stabilization of mutant protein, and to identify the sites of interaction between alpha AG98R and the peptide chaperone. Methods: Wild-type alpha A-crystallin (alpha AWT) and recombinant mutant alpha AG98R were purified from Escherichia coli BL21(DE3) pLysS cells. The zeta potential values of alpha A-crystallins in the presence or absence of alpha A-minichaperone and purified protein-peptide complexes were estimated in a zeta potential analyzer. Potential regions within alpha AG98R that bind the alpha A-minichaperone were investigated by incubating the protein with a photoactivable minichaperone variant, followed by mass spectrometric analysis. Results: Binding of the alpha A-minichaperone to aggregation-prone alpha AG98R was accompanied by an increase in the zeta potential from -15.19 +/- 0.870 mV corresponding to aAG98R alone to -28.64 +/- 1.640 mV for the purified complex. Mass spectrometric analysis identified (1)MDVTIQHPWFK(11), (13)TLGPFYPSR(21), (55)TVLDSGISEVR(65), and (EFHRR117)-E-113 as the alpha A-minichaperone-binding regions in alpha AG98R. The results suggest the involvement of the N-terminal region and the alpha-crystallin domain in the peptide-mediated stabilization of aAG98R. Conclusions: The alpha A-crystallin-derived minichaperone stabilizes alpha AG98R by compensating its lost surface charge. Methods for increasing the zeta potential of aggregating proteins can be a potential approach for therapy to protein aggregation diseases.

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