|Budget Amount *help
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
A growing number of evidence elucidates that multiple interactions between distinct molecular chaperones and their cofactors are essential to accomplish proper protein proper protein folding in the cell. We have demonstrated that two mammalian DnaJ homologs, Hsp4O and Hdj2, are close in the activity of stimulating the ATPase activity of Hsc7O and cooperating with Hsc7O to prevent protein aggregation. In contrast, Hsp4O could not suppress irreversible aggregation of chemically denatured rhodanese upon dilution, while Hdj2 significantly prevented the aggregation of rhodanese.
We previously reported that Hsp9O captured firefly luciferase during thermal inactivation, maintaining it in a folding-competent state. The activity of luciferase was remarkably restored by addition of rabbit reticulocyte lysate (RL). In this research, it has been demonstrated that Hsc7O and Hsp4O are more effective for refolding of luciferase than Hsc7O and Hdj2, while other unidentified factor in RL is inevitably required in addition. To elucidate the molecular identity of this factor, we attempted to purify the active component contained in RL enabling the reactivation of luciferase captured by Hsp9O in the presence of Hsc7O and Hsp4O.We showed that effective refolding requires PA28, an activator of the 20S proteasome, as a new cofactor working in concert with Hsc7O and Hsp4O.Purified Hsc7O, Hsp4O and PA28 were necessary and sufficient to reconstitute Hsp9O-initiated retolding of luciferase. The kinetics of substrate binding suggested that PA28 may be the physical link between the Hsp9O-dependent capture of unfolded proteins and the Hsc7O-and ATP-dependent refolding process.