Temperature dependent chaperone‐like activity of alpha‐crystallin

Raman, Bakthisaran; Tangirala, Ramakrishna; Rao, Ch. Mohan

doi:10.1016/0014-5793(95)00440-k

Cited by 153 publications

(136 citation statements)

References 41 publications

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“…␣A-and ␣B-crystallin homoaggregates were generated by dialyzing individual crystallins from 8 M urea against 50 mM Tris-HCl buffer, pH 7.4, 100 mM NaCl. Such a slow refolding by removing the denaturant by dialysis (29) or rapidly by dilution (18) results in native ␣-crystallin. The molecular mass of the refolded ␣-crystallin, however, may vary depending on conditions like ionic strength, pH, and temperature (30).…”

Section: Resultsmentioning

confidence: 99%

“…Isolation and Purification of ␣-Crystallin-Calf lens ␣-crystallin was isolated and purified as described earlier (18). The fractions corresponding to ␣-crystallin were pooled and concentrated at 4°C using an Amicon ultrafiltration unit with an M r 30,000 cutoff.…”

Section: Methodsmentioning

confidence: 99%

“…To address the mechanistic aspects of the function of ␣-crystallin, we have used a nonthermal aggregation system and found that the chaperone-like activity of ␣-crystallin is temperature-dependent (17). Our studies with photoaggregation of ␥-crystallin (17), thermal aggregation of ␤-crystallin, and DTT 1 -induced aggregation of insulin (18) together with the rapid refolding of crystallins (19) and the role of ␣-crystallin in these processes resulted in a hypothesis that sheds some light on the chaperone-like activity of ␣-crystallin. These studies show that ␣-crystallin prevents the aggregation of nonnative structures by providing appropriately placed hydrophobic surfaces.…”

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confidence: 94%

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Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Datta

Rao

1999

Journal of Biological Chemistry

103

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␣-Crystallin, a heteromultimeric protein made up of ␣A-and ␣B-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of ␣-crystallin can enhance its chaperone-like activity severalfold. The two subunits of ␣-crystallin have extensive sequence homology and individually display chaperonelike activity. We have investigated the chaperone-like activity of ␣A-and ␣B-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, ␣B-crystallin shows significant protective ability even at subphysiological temperatures, at which ␣A-crystallin or heteromultimeric ␣-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of ␤ L -crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quenching by acrylamide shows that the tryptophans in ␣B-crystallin are more accessible than the lone tryptophan in ␣A-crystallin even at 25°C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on ␣B-crystallin. Circular dichroism studies show some tertiary structural changes in ␣A-crystallin above 50°C. ␣B-crystallin, on the other hand, shows significant alteration of tertiary structure by 45°C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, ␣B-crystallin is much more sensitive to temperaturedependent structural perturbation than ␣A-or ␣-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of ␣-crystallin and indicate different roles for the two proteins both in ␣-crystallin heteroaggregate and as separate proteins under stress conditions. ␣-Crystallin is a major protein of the mammalian lens and constitutes as much as 50% of its dry weight. Studies over the past few years have shown that ␣-crystallin is expressed in several nonlenticular tissues such as heart, brain, and kidney, and its expression is enhanced severalfold during stress and disease conditions (1-6). ␣-Crystallin is shown to have homology with small heat shock proteins (7-10). Horwitz (11) shows that ␣-crystallin can prevent the thermal aggregation of ␤-and ␥-crystallins and a few other proteins like a molecular chaperone. Demonstration of chaperone-like activity of ␣-crystallin has provided an excellent opportunity to investigate the mechanistic aspects of chaperone function in general and the role of ␣-crystallin under stress conditions in particular. It is possible that, in the lens, ␣-crystallin may chaperone the formation of the transparent and appropriately ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 94%

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Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Datta

Rao

1999

Journal of Biological Chemistry

103

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“…Insulin requires 5-6 times its own concentration (w/w) of -crystallin for complete prevention. 9,33 It is true that thermal assay usually requires relatively less amount of -crystallin because of its higher activity at high temperature, 10,22,34 but it still requires approximately a ratio of 1:1 (w/w) between substrate and -crystallin for nearly full protection (data not shown). However, nearly complete prevention of aggregation of -and -crystallin by substoichiometric amount of -crystallin clearly indicates a different type of interaction and specificity.…”

Section: Discussionmentioning

confidence: 99%

Differential recognition of natural and nonnatural substrate by molecular chaperone α‐crystallin—A subunit exchange study

Biswas

Das

2006

Biopolymers

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Abstractα‐Crystallin is a molecular chaperone that recognizes proteins substrates in stress. It binds to the unstable conformer of a large variety of related or unrelated substrates and thus prevents them aggregating and holds them in a folding competent state. In this article, we have tried to critically analyze, from experimental point of view, whether α‐crystallin has any preference for its natural substrates compared to the nonnatural one. Our results clearly show that α‐crystallin is exceptionally active and sensitive in preventing aggregation of its natural substrates and can fully prevent such an aggregation in a substoichiometric ratio, but nonnatural substrates require a considerably higher amount of α‐crystallin. Using suitable fluorescent‐labeled α‐crystallins and performing fluorescence resonance energy transfer experiments, we were able to determine the subunit exchange kinetics between the α‐crystallin oligomers. It was found that while α‐crystallin was bound to its natural substrate, the rate of subunit exchange was slightly decreased. But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was observed. Nonnatural substrate was found to create higher activation energy barrier for the subunit exchange reaction compared to the native substrates. Similarities in major β‐sheet structure of both α‐crystallin and its natural substrates may be the reason for the preference in molecular recognition in comparison with the nonnatural substrate. © 2006 Wiley Periodicals, Inc. Biopolymers 85:189–197, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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“…Whereas hydrophobic interactions are involved in the chaperone action of ␣-crystallin, the specific mechanism of this interaction is not known (33)(34)(35). ␣-Crystallin does not bind to unfolded proteins or compact folded proteins (35) or to chemically modified, molten-globule states that are stable in solution and do not aggregate (31,36).…”

Section: Discussionmentioning

confidence: 99%

The Molecular Chaperone, α-Crystallin, Inhibits Amyloid Formation by Apolipoprotein C-II

Hatters

Lindner

Carver

et al. 2001

Journal of Biological Chemistry

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Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, ␣-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of ␣-crystallin (1-10 g/ml). ␣-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of ␣-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced ␣-lactalbumin. Two pieces of evidence suggest that ␣-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between ␣-crystallin and unstructured monomeric apoC-II. Second, the addition of ␣-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that ␣-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that ␣-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, ␣-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

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Temperature dependent chaperone‐like activity of alpha‐crystallin

Cited by 153 publications

References 41 publications

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Differential recognition of natural and nonnatural substrate by molecular chaperone α‐crystallin—A subunit exchange study

The Molecular Chaperone, α-Crystallin, Inhibits Amyloid Formation by Apolipoprotein C-II

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