Chaperone-like Activity Of Alpha-crystallin Research Articles

Subunit Mobility and the Chaperone Activity of Recombinant aB-Crystallin

The comparison of the chaperone-like activity of native and covalently cross-linked human αB-crystallins has confirmed the important role of the subunit mobility in the chaperoning mechanism. Our data clearly demonstrate that the chaperone-like activity of α-crystallin is not only a surface phenomenon as was suggested by some researchers.

Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, alphaB-(1-18) (MDIAIHHPWIRRPFFPFH) and betaA3/A1-(59-74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, gammaS-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact alpha-, beta-, and gamma-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of alphaB-(1-18) and betaA3/A1-(59-74) peptides increased the scattering of light by beta- and gamma-crystallin and alcohol dehydrogenase. The ability of alpha-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with alphaB-(1-18) and betaA3/A1-(59-74) peptides, whereas gammaS peptide had no effect on chaperone-like activity of alpha-crystallin. The betaA3/A1-(59-74 peptide caused a 5.64-fold increase in alphaB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in alphaB-(1-18) and betaA3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.

Chaperone‐like activity and hydrophobicity of α‐crystallin

alpha-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, alphaA- and alphaB-crystallins. Numerous studies have demonstrated that alpha-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 alpha-crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. alpha-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 alpha-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 alpha-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 alpha-crystallin, the kind of evaluation done for the first time.

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.

Chaperone-like activity of alpha-crystallin and other small heat shock proteins.

Small heat shock proteins (sHsps) are a large family of proteins with monomeric molecular weight of 12-43 kDa, present within the prokaryotic and eukariotic cell as large oligomeric complexes, ranging in size from 200-800 kDa. Unlike the high molecular weight Hsps, which are involved in protein folding in vivo, under normal conditions, sHsps play an important role in protecting organism from stress. SHsps share an evolutionarily conserved sequence of 80-100 amino acids, located in the C-terminal region, and called "alpha-crystallin domain"; its role in subunits interactions has been recently underlined by site-directed spin labeling studies and by fluorescence resonance energy transfer data. The N-terminal region, preceding the alpha-crystallin domain, is variable in length and amino acid sequence, contributing to structural diversity between different sHsps and having a role in multimerization. The alpha-Crystallin domain is followed by C-terminal extension, a polar structure, involved in protein solubility, which share no sequence homology. Expression of sHsps is induced in response to various kinds of stress including heat shock, oxidative stress, osmostress, or ischemia, but some sHsps are expressed constitutively under physiological conditions. In vitro, sHsps selectively bind and stabilize proteins and prevent their aggregation at elevated temperatures in an ATP-independent way and protect enzymes against heat-induced inactivation. Our own studies focused on the chaperone-like activity of alpha-crystallin, the major protein component of vertebrate lens, using another system than heat-induced aggregation. Our data demonstrated that alpha-crystallin specifically protects enzymes against inactivation by different posttranslational modifications such as glycation, carbamylation and aldehyde binding, and also reactivates GuHCl-denatured enzymes. Complex formation between alpha-crystallin and the denatured enzymes, was suggested as a mechanism of protection.

Alpha-crystallin does not require temperature activation for its chaperone-like activity.

alpha-crystallin acts as a molecular chaperone by preventing the aggregation of proteins. Although the mechanism for this activity is not understood there is a proposition that temperature activation at or above 30 degrees C of alpha-crystallin is an absolute requirement, thereby suggesting a conformational transition as a trigger for the activity. In an attempt to unravel the putative temperature-activity relationship, the chaperone-like activity of alpha-crystallin was studied at a number of temperatures above and below 30 degrees C. Chaperone activity was monitored against aggregation of the insulin-B chain induced by cleavage of disulfide bond of insulin and also against photo-aggregation of gamma-crystallin. Contrary to the above notion, the results indicate that alpha-crystallin does not require thermal activation for its chaperone function and that it can efficiently function as a molecular chaperone even at temperatures below the previously reported transition temperature.

Chaperone-like Activity and Temperature-induced Structural Changes of α-Crystallin

alpha-Crystallin is known to exhibit chaperone-like activity. We have studied its chaperone-like activity toward the aggregation of betaL-crystallin upon refolding of this protein from its unfolded state in guanidinium chloride. The chaperone-like activity of alpha-crystallin is less pronounced below 30 degrees C and is enhanced above this temperature. The plot of percentage protection as a function of temperature shows two transitions; one at 30 degrees C and another at around 55 degrees C. We have performed steady state fluorescence, fluorescence polarization, fluorescence quenching, circular dichroism, sedimentation analysis, and gel filtration chromatography to probe the temperature-induced structural changes of alpha-crystallin. Our results show that at above 50 degrees C, alpha-crystallin undergoes a transition to a multimeric molten globule-like state. Above 30 degrees C, a minor but detectable perturbation in its tertiary structure occurs that might lead to the observed exposure of its hydrophobic surfaces. These results support our earlier hypothesis that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or reorganized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. It is possible that a structural alteration induced by temperature forms a part of the general mechanism of chaperone function, because they are required to function more effectively at nonpermissible temperatures.

Rapid Refolding Studies on the Chaperone-like α-Crystallin: EFFECT OF α-CRYSTALLIN ON REFOLDING OF β- AND γ-CRYSTALLINS

alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and gamma-crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and gamma-crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and gamma-crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and gamma-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.

Chaperone-like activity of alpha-crystallin. The effect of NADPH on its interaction with zeta-crystallin.

alpha-Crystallin, a major structural protein of the ocular lens of vertebrates, has been characterized recently as a molecular chaperone (Horwitz, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10449-10453 and Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520). While alpha-crystallins prevent the aggregation of various proteins denatured by heat or chaotropic agents, neither the mode of interaction between target proteins and alpha-crystallin nor the specific conformational requirements, if any, of the target protein are known. Here, we demonstrate that the ability of alpha-crystallin to prevent thermally induced aggregation of zeta-crystallin/NADPH:quinone oxidoreductase, an abundant crystallin of guinea pigs and camelids, is strongly dependent on the presence of the obligate cofactor (NADPH) of the target enzyme. zeta-crystallin in the absence of NADPH is readily aggregated at 41 degrees C, and alpha-crystallin added at a 1:1 (w/w) ratio offers very little protection. In contrast, in the presence of NADPH zeta-crystallin remains stable to 45 degrees C and with the addition of alpha-crystallin (1:1 (w/w)) is protected from aggregation even at 55 degrees C. Cibacron blue 3GA, a nonmetabolized pyridine nucleotide analog, which has very high binding affinity to zeta-crystallin had similar effects. NADH and NAD+, which are not bound by zeta-crystallin, had no such effect. Complex formation between alpha-crystallin and non-native zeta-crystallin was demonstrated in the presence of either cibacron blue 3GA or NADPH. Circular dichroism spectroscopy of zeta-crystallin in the presence and absence of NADPH or cibacron blue indicated that nucleotide binding was accompanied by a change in the protein's aromatic amino acid environment but that the secondary structure was unaffected. The data suggest that subtle change in the conformation of denaturing proteins can markedly affect the ability of alpha-crystallin to protect them from aggregation.