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.
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 microm radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion 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 O(2)() 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 microm, 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.
Billecke
Scott S
SS
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Bender
Andrew T
AT
Kanelakis
Kimon C
KC
Murphy
Patrick J M
PJ
Lowe
Ezra R
ER
Kamada
Yasuhiko
Y
Pratt
William B
WB
Osawa
Yoichi
Y
D000818:Animals; D016227:Benzoquinones; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006418:Heme / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D016877:Oxidants / Q000378:metabolism*; D011485:Protein Binding; D011809:Quinones / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D016877:Oxidants; D011809:Quinones; D011994:Recombinant Proteins; D006418:Heme; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C035359:monorden; C001277:geldanamycin