Mammalian HSP60 is a major target for an immunosuppressant mizoribine.
It has been reported that immunosuppressant cyclosporin A or FK506 binds to immunophilins in the cell and that these immunophilins make a complex with molecular chaperones HSP70 or HSP90. Although mizoribine has been used clinically as an immunosuppressant, immunophilins of the agent have not yet been fully understood. We investigated their specific binding proteins using mizoribine affinity column chromatography and porcine kidney cytosols. By increasing mizoribine in the eluant from the column, two major proteins (with molecular masses of 60 and 43 kDa) were detected by SDS-polyacrylamide gel electrophoresis. Based on the amino acid sequence analysis of these proteins, 60- and 43-kDa mizoribine-binding proteins were identified with HSP60 and cytosolic actin, respectively. A considerable amount of actin was also eluted from the affinity column by nucleotides, but a very low quantity of HSP60 was eluted under the same conditions. On the other hand, HSP60 was eluted as a major protein in the eluant that was eluted preferentially, with nucleotide followed by mizoribine. Actin was also detected in the eluant, but the quantity of the protein was very low. These results indicated that HSP60 has high affinity to mizoribine, and the interaction was also observed on surface plasmon resonance analysis. Although HSP60 or GroE facilitated refolding of citrate synthase in vitro, mizoribine interfered with the chaperone activity of HSP60. On different types of mizoribine affinity columns, HSP60 or actin recognized the NH(2) group of mizoribine, and this group may be a functional group of the agent.
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
Komatsuda
A
A
Wakui
H
H
Miura
A B
AB
Tashima
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D018834:Chaperonin 60 / Q000187:drug effects* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism; D018832:Molecular Chaperones / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D011485:Protein Binding; D012263:Ribonucleosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013552:Swine; D013997:Time Factors
D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; D007166:Immunosuppressive Agents; D018832:Molecular Chaperones; D009711:Nucleotides; D012263:Ribonucleosides; C010052:mizoribine; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase