Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. ␣ 1 -Antichymotrypsin (ACT) and ␣ 1 -antitrypsin (␣ 1 -AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp ␣-crystallin was unable to prevent the nucleation-independent aggregation of ␣ 1 -AT, whereas ␣-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight ␣-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4 -Dianilino-1,1 -binaphthyl-5,5 -disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that ␣-crystallin interacts specifically with a non-native conformation of ACT. The finding that ␣-crystallin does not interact with ␣ 1 -AT under these conditions suggests that ␣-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of ␣-crystallin and other sHsps.

Section: Discussionmentioning

confidence: 88%

The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, α-Crystallin, Indicates a Nucleation-dependent Specificity

Devlin

Carver

Bottomley

2003

“…In nonlenticular tissues, the role of small heat shock proteins may be to maintain substrate proteins in a folding-competent state (15,16). Although the mechanism of the chaperone action of ␣-crystallin is at present not fully understood, it was shown that ␣-crystallin specifically recognizes aggregation-prone nonnative structures that occur early on the denaturation pathway of proteins (17)(18)(19). Furthermore, a number of studies suggest that the chaperone activity of the protein is dependent on the presence of surface-exposed hydrophobic patches (11, 20 -25).…”

mentioning

confidence: 99%

Temperature-dependent Chaperone Activity and Structural Properties of Human αA- and αB-crystallins

Reddy¹,

Das²,

Petrash³

et al. 2000

158

175

The chaperone activity and biophysical properties of recombinant human ␣A-and ␣B-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of ␣A-crystallin markedly improves with an increase in temperature, the activity of ␣B homopolymer appears to change very little upon heating. Compared with ␣B-crystallin, the ␣A-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the ␣B homopolymer appears to be modestly (two times or less) more potent chaperone than ␣A homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, ␣A-and ␣B-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, ␣A-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for ␣B-crystallin. The correlation between temperaturedependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of ␣-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of ␣-crystallin.␣-Crystallin, a major lens protein, plays an important role in maintaining the transparency and refractive properties of the eye lens (1-3). The lenticular ␣-crystallin consists of two 20-kDa polypeptide chains, ␣A and ␣B, that share about 60% sequence homology. These chains self-associate, forming large oligomeric complexes that contain 30 -50 subunits at the ␣A to ␣B ratio of approximately 3:1. Although the high resolution structure of ␣-crystallin is currently not known, recent cryoelectron microscopy data shows that the recombinant ␣B-

“…Otherwise, they differ in sequence, although there are conserved residues within variable regions that may be essential to their chaperone-like activity (15,19,20). The ATP-independent chaperonelike activity of these proteins is seemingly more directed to preventing protein aggregation early in denaturation, rather than the active refolding of compromised proteins (19,(21)(22)(23)(24)(25). This is an especially important characteristic of the ␣-crystallins, major proteins of the lens which limit aggregation of other eye crystallins and maintain lens transparency (23)(24)(25).…”

mentioning

confidence: 99%

“…The ATP-independent chaperonelike activity of these proteins is seemingly more directed to preventing protein aggregation early in denaturation, rather than the active refolding of compromised proteins (19,(21)(22)(23)(24)(25). This is an especially important characteristic of the ␣-crystallins, major proteins of the lens which limit aggregation of other eye crystallins and maintain lens transparency (23)(24)(25). The ␣-crystallins, and especially ␣B-crystallin, are also found in nonlenticular tissues (26 -28), where their synthesis is induced by stress (24,(27)(28)(29)(30)(31).…”

mentioning

confidence: 99%

Molecular Characterization of a Small Heat Shock/α-Crystallin Protein in Encysted Artemia Embryos

Liang¹,

Amons²,

Clegg³

et al. 1997

Molecular chaperones protect cells during stress by limiting the denaturation/aggregation of proteins and facilitating their renaturation. In this context, brine shrimp embryos can endure a wide variety of stressful conditions, including temperature extremes, prolonged anoxia, and desiccation, thus encountering shortages of both energy (ATP) and water. How the embryos survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embryos. p26 is the first representative of the small heat shock/␣-crystallin family from crustaceans to be sequenced, and it possesses the conserved ␣-crystallin domain characteristic of these proteins. The secondary structure of this domain was predicted to consist predominantly of ␤-pleated sheet, and it appeared to lack regions of ␣-helix. Unique properties of the nonconserved amino terminus, which showed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/␣-crystallin protein, and it is hydrophilic, a common attribute of this region. Site-specific differences between amino acids from p26 and other small heat shock/␣-crystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene family for p26, whereas two size classes of p26 mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the larger probably representing nonprocessed transcripts. Examination of immunofluorescently stained samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and that it resides within discrete compartments of this organelle. The results in this paper demonstrate clearly that p26 is a novel member of the small heat shock/␣-crystallin family of proteins. These data, in concert with its restriction to embryos undergoing oviparous development, suggest that p26 functions as a molecular chaperone during exposure to stress, perhaps able to limit protein degradation and thus ensure a ready supply of functional proteins when growth is reinitiated.