A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90.
The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N- and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the N-domain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS/PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants from the control column on SDS/PAGE. These results indicated that CDDP has a high affinity for HSP90. On CD spectrum analysis, the binding of CDDP to HSP90 resulted in a conformational change in the protein. Although HSP90 inhibited the aggregation of citrate synthase as a molecular chaperone in vitro, the activity was suppressed almost completely in the presence of CDDP. Mg/ATP has an influence on the chaperone activity to some extent. The CDDP binding region of HSP90 is near the C-terminal which is quite different from the GA-binding site. Our results suggest that the chaperone activity of HSP90 may be inhibited by the binding of CDDP or GA by different mechanisms.
Itoh
H
H
Department of Biochemistry, Akita University School of Medicine, 1-1-1 Hondo, Akita City 010-8543, Japan. hideaki@med.akita-u.ac.jp
Ogura
M
M
Komatsuda
A
A
Wakui
H
H
Miura
A B
AB
Tashima
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D002945:Cisplatin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011809:Quinones / Q000494:pharmacology
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D010446:Peptide Fragments; D011809:Quinones; D000255:Adenosine Triphosphate; D002945:Cisplatin; C001277:geldanamycin