S100A1 Is a Novel Molecular Chaperone and a Member of the Hsp70/Hsp90 Multichaperone Complex

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 ␤-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.

Interactions of S100A2 and S100A6 with the Tetratricopeptide Repeat Proteins, Hsp90/Hsp70-organizing Protein and Kinesin Light Chain

“…Expression and Purification of Recombinant Proteins-All recombinant S100 proteins (S100B, A1, A2, A4, A6, A10, A11, A12, and A13) were expressed in a tag-free fashion and prepared as described previously (5,26). Briefly, S100 proteins expressed in BL21 (DE3) were extracted using TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and purified by phenyl-Sepharose column chromatography.…”

“…Recently, we identified S100A1 to be a new member of the multichaperone complex and a potent molecular chaperone (5). To identify additional possible chaperone-interacting S100 proteins, we examined their interaction with the co-chaperone Hsp70/Hsp90-organizing protein (Hop).…”

“…Family-Previously, we showed that S100A1 interacts with Hsp90, Hsp70, FK506-binding protein 52 (FKBP52), and cyclophilin40 (CyP40) (5). To further study the interaction of S100 proteins with molecular chaperones, we performed an in vitro binding assay using GST-Hop in the presence of 1 mM CaCl 2 or 1 mM EGTA (Fig.…”

Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN-45 gastric carcinoma cells

“…Histidine-tagged PP5 (His-PP5) protein was expressed and purified according to the manufacturer's instructions. S100A1 and S100A4 proteins were expressed and purified as previously described (22,23). Purified recombinant S100A4 proteins were diluted (0.5 mg/ml) and maintained at -30˚C in a freezer for 3 months (air-oxidized).…”

Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity

“…Histidine-tagged PP5 (His-PP5) protein was expressed and purified according to the manufacturer's protocol. S100 proteins (S100A1, S100A2, S100A6, S100B, and S100P) were expressed and purified as described previously (Yamashita et al 1999;Okada et al 2004). Air-oxidized S100 proteins were prepared by storing the recombinant S100 proteins (0.5 mg/mL) at -30°C for 3 months.…”