M Satish Kumar

Chaperone-like activity and hydrophobicity of α-crystallin

α‐Crystallin, a prominent member of small heat shock protein (sHsp) family and a major structural protein of the eye lens is a large polydisperse oligomer of two isoforms, αA‐ and αB‐crystallins. Numerous studies have demonstrated that α‐crystallin functions like a molecular chaperone in preventing the aggregation of various proteins under a wide range of stress conditions. The molecular chaperone function of α‐crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. α‐Crystallin selectively interacts with non‐native proteins thereby preventing them from aggregation and helps maintain them in a folding competent state. It has been proposed and generally accepted that α‐crystallin suppresses the aggregation of other proteins through the interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded substrate protein. However, a quantifiable relationship between hydrophobicity and chaperone‐like activity remains a matter to be concerned about. On an attentive review of studies on α‐crystallin chaperone‐like activity, particularly the studies that have direct or indirect implications to hydrophobicity and chaperone‐like activity, we found several instances wherein the correlation between hydrophobicity and its chaperone‐like activity is paradoxical. We thus attempted to provide an overview on the role of hydrophobicity in chaperone‐like activity of α‐crystallin, the kind of evaluation done for the first time. iubmb Life, 58: 632 ‐ 641, 2006

Effect of glycation on α-crystallin structure and chaperone-like function

The chaperone-like activity of α-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of α-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of α-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of α-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of α-crystallin. Modification of α-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [Nϵ-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of α-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of α-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, α-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of α-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describes the effects of glycation on the structure and chaperone-like activity of α-crystallin.

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.

αB-crystallin-assisted reactivation of glucose-6-phosphate dehydrogenase upon refolding

Alphab-crystallin, a small heat-shock protein has been shown to prevent the aggregation of other proteins under various stress conditions. We have investigated the role of alphaB-crystallin in the reactivation of denaturant [GdmCl (guanidinium chloride)]-inactivated G6PD (glucose-6-phosphate dehydrogenase). Studies indicate that unfolding and inactivation of G6PD by GdmCl proceeds via formation of a molten globule-like state at low concentrations of GdmCl, which was characterized by having maximum surface hydrophobicity and no catalytic activity. At high concentrations of GdmCl, G6PD was completely unfolded, which upon dilution-induced refolding yielding 35% of original activity. In contrast, no activity was recovered when G6PD was refolded from a molten globule-like state. Interestingly, refolding of completely unfolded G6PD in the presence of alphaB-crystallin resulted in 70% gain of the original activity, indicating that alphaB-crystallin assisted in enhanced refolding of G6PD. Intriguingly, alphaB-crystallin was unable to reactivate G6PD from a molten globule-like state. Size-exclusion chromatography data indicate that alphaB-crystallin-assisted reactivation of completely unfolded G6PD is concomitant with the restoration of the native structure of G6PD. Nonetheless, alphaB-crystallin failed to reactivate G6PD from preformed aggregates. Moreover, methylglyoxal-modified alpha-crystallin, which occurs in aged and diabetic cataract lenses, was less efficient in the reactivation of denaturant inactivated G6PD. Diminished chaperone-like activity of alpha-crystallin due to post-translational modifications may thus result in the accumulation of aggregated/inactivated proteins.