Chaperone-like Activity of Tubulin

Self Cite

110

160

ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone ␣-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of ␣-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4 -Dianilino-1,1 -binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on ␣-crystallin, but no appreciable enhancement of the same was observed for the substrate protein ␥-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mM concentration stabilizes the ␣-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone ␣-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to ␣-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5 -O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of ␣-crystallin in vivo is discussed.␣-Crystallin is the major protein component of the vertebrate eye lens and is composed of two subunits, ␣A and ␣B, 20 kDa each having 57% sequence homology between them. Both subunits associate to form a large oligomer with an average molecular mass of 800 kDa. It is a key member of the small heat shock protein (sHSP) 1 superfamily having a conserved core

Section: Discussionsupporting

confidence: 85%

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Biswas

Das

2004

Self Cite

110

160

“…However, considering the known three-dimensional structures of spectrins (three-helix bundle and rodlike shape) and that of the chaperones (mostly globular), it is unlikely that spectrin will have structural homology with the chaperones. This has been also observed with the tubulin, which has a tubular structure and is known to have chaperone like activity (17,18).…”

Section: Discussionmentioning

confidence: 57%

“…Among the chaperone proteins not belonging to the Hsp family, ␣-crystallin was first shown to have chaperone-like function in suppressing aggregation of various proteins by heat or by using other agents that induce aggregation (16). Similarly for tubulin, a ubiquitous cytoskeletal protein and the building block unit of the microtubule assembly, it was observed that the protein could also suppress both thermal and nonthermal aggregation of a number of unrelated proteins, and the negatively charged C-terminal tail plays a crucial role for its chaperone like activity (17,18).…”

mentioning

confidence: 99%

Chaperone Activity and Prodan Binding at the Self-associating Domain of Erythroid Spectrin

Bhattacharyya

Ray²,

Bhattacharya

et al. 2004

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25°C and that of alcohol dehydrogenase at 50°C. The dimeric spectrin and the two subunits, ␣-spectrin and ␤-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes ␣-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)-naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in ␣-and ␤-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in ␣-spectrin near the N terminus with human Hsp90 and in ␤-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of ␣-spectrin near the N terminus and the ␤-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.

“…The N-terminal Region Mediates Substrate Protein Binding-Small molecular chaperones, such as small heat shock proteins, ␣-crystallin, tubulin, and clusterin, prevent protein precipitation by forming soluble HMW complexes (27,(32)(33)(34)(35)(36)(37)(38)(39)(40). We first confirmed that ␣-synuclein also acts in this way to prevent protein precipitation, and then investigated which re- gion of ␣-synuclein was critical for substrate protein binding (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…by forming high molecular weight (HMW) complexes with partially unfolded substrate proteins (27,(32)(33)(34)(35)(36)(37)(38)(39)(40). However, alone sHSPs do not have the ability to protect enzymes from thermal inactivation or to promote their functional refolding after de-naturation (31,39,41,42), although a few exceptional cases with marginal effects have been reported (30,33,(43)(44)(45)(46). sHSPs have, therefore, been classified as "junior chaperones" (47).…”

mentioning

confidence: 99%

Distinct Roles of the N-terminal-binding Domain and the C-terminal-solubilizing Domain of α-Synuclein, a Molecular Chaperone

Park

Jung²,

Kim³

et al. 2002

113

␣-Synuclein, an acidic neuronal protein of 140 amino acids, is extremely heat-resistant and is natively unfolded. Recent studies have demonstrated that ␣-synuclein has chaperone activity both in vitro and in vivo, and that this activity is lost upon removing its C-terminal acidic tail. However, the detailed mechanism of the chaperone action of ␣-synuclein remains unknown. In this study, we investigated the molecular mechanism of the chaperone action of ␣-synuclein by analyzing the roles of its N-terminal and C-terminal domains. The N-terminal domain (residues 1-95) was found to bind to substrate proteins to form high molecular weight complexes, whereas the C-terminal acidic tail (residues 96 -140) appears to be primarily involved in solubilizing the high molecular weight complexes. Because the substrate-binding domain and the solubilizing domain for chaperone function are well separated in ␣-synuclein, the N-terminal-binding domain can be substituted by other proteins or peptides. Interestingly, the resultant engineered chaperone proteins appeared to display differential efficiency and specificity in terms of the chaperone function, which depended upon the nature of the binding domain. This finding implies that the C-terminal acidic tail of ␣-synuclein can be fused with other proteins or peptides to engineer synthetic chaperones for specific purposes.