Epolactaene Binds Human Hsp60 Cys442 Resulting in the Inhibition of Chaperone Activity

Overview

Journal Biochem J

Specialty Biochemistry

Date 2004 Dec 18

PMID 15603555

Citations 30

Authors

Yoko Nagumo

Hideaki Kakeya

Mitsuru Shoji

Yujiro Hayashi

Naoshi Dohmae

Hiroyuki Osada

Affiliations

Soon will be listed here.

Abstract

Epolactaene is a microbial metabolite isolated from Penicillium sp., from which we synthesized its derivative ETB (epolactaene tertiary butyl ester). In the present paper, we report on the identification of the binding proteins of epolactaene/ETB, and the results of our investigation into its inhibitory mechanism. Using biotin-labelled derivatives of epolactaene/ETB, human Hsp (heat-shock protein) 60 was identified as a binding protein of epolactaene/ETB in vitro as well as in situ. In addition, we found that Hsp60 pre-incubated with epolactaene/ETB lost its chaperone activity. The in vitro binding study showed that biotin-conjugated epolactaene/ETB covalently binds to Hsp60. In order to investigate the binding site, binding experiments with alanine mutants of Hsp60 cysteine residues were conducted. As a result, it was suggested that Cys442 is responsible for the covalent binding with biotin-conjugated epolactaene/ETB. Furthermore, the replacement of Hsp60 Cys442 with an alanine residue renders the chaperone activity resistant to ETB inhibition, while the alanine replacement of other cysteine residues do not. These results indicate that this cysteine residue is alkylated by ETB, leading to Hsp60 inactivation.

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References

Kakeya H, Onozawa C, Sato M, Arai K, Osada H . Neuritogenic effect of epolactaene derivatives on human neuroblastoma cells which lack high-affinity nerve growth factor receptors. J Med Chem. 1997; 40(4):391-4. DOI: 10.1021/jm960719a. View

Xanthoudakis S, Roy S, Rasper D, Hennessey T, Aubin Y, Cassady R . Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis. EMBO J. 1999; 18(8):2049-56. PMC: 1171289. DOI: 10.1093/emboj/18.8.2049. View

Xu Z, Horwich A, SIGLER P . The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 1997; 388(6644):741-50. DOI: 10.1038/41944. View

Viitanen P, Lorimer G, Bergmeier W, Weiss C, Kessel M, Goloubinoff P . Purification of mammalian mitochondrial chaperonin 60 through in vitro reconstitution of active oligomers. Methods Enzymol. 1998; 290:203-17. DOI: 10.1016/s0076-6879(98)90020-9. View

Itoh H, Komatsuda A, Wakui H, Miura A, Tashima Y . Mammalian HSP60 is a major target for an immunosuppressant mizoribine. J Biol Chem. 1999; 274(49):35147-51. DOI: 10.1074/jbc.274.49.35147. View

Kol A, Lichtman A, Finberg R, Libby P . Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol. 1999; 164(1):13-7. DOI: 10.4049/jimmunol.164.1.13. View

Mizushina Y, Kobayashi S, Kuramochi K, Nagata S, Sugawara F, Sakaguchi K . Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II. Biochem Biophys Res Commun. 2000; 273(2):784-8. DOI: 10.1006/bbrc.2000.3007. View

Sawano A, Miyawaki A . Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 2000; 28(16):E78. PMC: 108465. DOI: 10.1093/nar/28.16.e78. View

Ki S, Ishigami K, Kitahara T, Kasahara K, Yoshida M, Horinouchi S . Radicicol binds and inhibits mammalian ATP citrate lyase. J Biol Chem. 2000; 275(50):39231-6. DOI: 10.1074/jbc.M006192200. View

10.

Thirumalai D, Lorimer G . Chaperonin-mediated protein folding. Annu Rev Biophys Biomol Struct. 2001; 30:245-69. DOI: 10.1146/annurev.biophys.30.1.245. View

11.

Viitanen P, Weiss C, Sharkia R, Greenberg A, Niv A, Lustig A . The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60. Eur J Biochem. 2001; 268(12):3465-72. DOI: 10.1046/j.1432-1327.2001.02243.x. View

12.

Barazi H, Zhou L, Templeton N, Krutzsch H, Roberts D . Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation. Cancer Res. 2002; 62(5):1541-8. View

13.

Nakai J, Kawada K, Nagata S, Kuramochi K, Uchiro H, Kobayashi S . A novel lipid compound, epolactaene, induces apoptosis: its action is modulated by its side chain structure. Biochim Biophys Acta. 2002; 1581(1-2):1-10. DOI: 10.1016/s1388-1981(01)00169-x. View

14.

Kirchhoff S, Gupta S, Knowlton A . Cytosolic heat shock protein 60, apoptosis, and myocardial injury. Circulation. 2002; 105(24):2899-904. DOI: 10.1161/01.cir.0000019403.35847.23. View

15.

Bethke K, Staib F, Distler M, Schmitt U, Jonuleit H, Enk A . Different efficiency of heat shock proteins (HSP) to activate human monocytes and dendritic cells: superiority of HSP60. J Immunol. 2002; 169(11):6141-8. DOI: 10.4049/jimmunol.169.11.6141. View

16.

Itoh H, Komatsuda A, Ohtani H, Wakui H, Imai H, Sawada K . Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration. Eur J Biochem. 2002; 269(23):5931-8. DOI: 10.1046/j.1432-1033.2002.03317.x. View

17.

Hayashi Y, Kanayama J, Yamaguchi J, Shoji M . Diastereoselective total synthesis of both enantiomers of epolactaene. J Org Chem. 2002; 67(26):9443-8. DOI: 10.1021/jo025641m. View

18.

Miyake Y, Kakeya H, Kataoka T, Osada H . Epoxycyclohexenone inhibits Fas-mediated apoptosis by blocking activation of pro-caspase-8 in the death-inducing signaling complex. J Biol Chem. 2003; 278(13):11213-20. DOI: 10.1074/jbc.M209610200. View

19.

Shan Y, Liu T, Su H, Samsamshariat A, Mestril R, Wang P . Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells. J Mol Cell Cardiol. 2003; 35(9):1135-43. DOI: 10.1016/s0022-2828(03)00229-3. View

20.

Nagumo Y, Kakeya H, Yamaguchi J, Uno T, Shoji M, Hayashi Y . Structure-activity relationships of epolactaene derivatives: structural requirements for inhibition of Hsp60 chaperone activity. Bioorg Med Chem Lett. 2004; 14(17):4425-9. DOI: 10.1016/j.bmcl.2004.06.054. View