Abstract
The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 degrees C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. alpha-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.
Highlights
The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species
Dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 °C, which is coupled with self-oligomerization of HSP90
Since HSP90 is known to function as a molecular chaperone, the oligomerization may be a consequence of recognizing substrate HSP90 by chaperone HSP90
Summary
The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. We found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages. If they are preheated at nonlethally high temperatures, they can survive the subsequent exposure to 45 °C [2, 3] These results were interpreted as indications that the acquisition of thermotolerance is attributed to the accumulation of stress proteins. Since the mode of action of HSP90 has not been fully investigated, its function as a molecular chaperone remains elusive It has been shown recently, that HSP90 functions as a molecular chaperone to prevent protein aggregation [24, 25] and assist refolding of denatured proteins [26]. The endoplasmic reticulum homologue of HSP90, Grp, was shown to mediate folding and assembly of immunoglobulin chains [27]
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