Abstract
Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.
Highlights
Calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain
To establish whether CaM or S100 proteins exhibited properties of molecular chaperones, we examined their interaction with the components of the chaperone complex and activities in the refolding of substrate proteins in vivo and in vitro
Hsp90, Glutathione S-transferase (GST)-truncated Hsp70, GST-FKBP52, and GST-CyP40 fusion proteins to confirm the interactions between CaM and the chaperone and cochaperone proteins described in previous reports [15,16,17,18] and exclude the possibility that these interactions were indirect or nonspecific
Summary
Heat shock protein; FKBP, FK506binding protein; CyP40, cyclophilin; Hop, Hsp organizing protein; CS, citrate synthase; CaM, calmodulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; SPR, surface plasmon resonance; OAA, oxaloacetic acid; CaM KII, calcium/calmodulindependent protein kinase II. Similar to Hsp and Hsp, cochaperone proteins such as FKBP52, CyP40, and p23, but not Hop, have been found to display chaperone activity [9, 10]. These proteins prevent the thermal aggregation of citrate synthase (CS) or refold a denatured -galactosidase in vitro [9, 10]. It has been known that CaM and S100 proteins can have differential effects on a common target protein [20, 21] These reports prompted us to investigate the functional role of S100 proteins in the multichaperone complex. We have identified S100A1 to be a potent molecular chaperone and a new member of the multichaperone complex
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