hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase: Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90

Scott S. Billecke, Andrew T. Bender, Kimon C. Kanelakis, Patrick J.M. Murphy, Ezra R. Lowe, Yasuhiko Kamada, William B. Pratt, Yoichi Osawa

研究成果査読

65 被引用数 (Scopus)

抄録

It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 μM radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme in. sertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic heme-binding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOS-dependent O2-production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 μM, geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of geldanamycin as a specific inhibitor of hsp90 in redox-active systems.

本文言語English
ページ(範囲)20504-20509
ページ数6
ジャーナルJournal of Biological Chemistry
277
23
DOI
出版ステータスPublished - 6月 7 2002
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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