Neuronal Nitric-oxide Synthase Is Regulated by the hsp90-based Chaperone System in Vivo

Abstract

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR⅐hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS⅐hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS⅐hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.Several transcription factors and protein kinases involved in signal transduction are recovered from cells in association with the ubiquitous heat shock protein hsp90 1 (for review, see Refs.1 and 2). These heterocomplexes with hsp90 are formed by a multicomponent chaperone machinery consisting of hsp90, hsp70, Hop, hsp40, p23, and probably also the hsp70-interacting protein Hip and the GrpE-like protein BAG-1 (for review, see Ref.3 and references therein). As first shown for the glucocorticoid receptor (GR) (4) and then for some other steroid receptors and the dioxin (Ah) receptor, association of the ligand binding domain (LBD) with hsp90 is required for the high affinity ligand binding conformation (1, 2). Complexing of the GR with hsp90 also opens up both thiol moieties (5) and trypsin cleavage sites (6, 7) in the LBD to attack by a thiol-derivatizing agent and the protease. These direct data, coupled with recent genetic observations (8), support the notion (9, 10) that the hsp90-based chaperone machinery directs an ATP-dependent partial unfolding of the receptor LBD, thus making the hydrophobic steroid-binding pocket accessible to steroid. The problem of providing access of ligands to hydrophobic binding sites situated in the interior of properly folded proteins is not unique to steroid and dioxin receptors. To test whether the hsp90-based chaperone machinery may play a more general role in opening up hydrophobic binding clefts, we have asked whether this system facilitates the...

Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON

“…Conceivably, the marked increases of CAPON and nNOS in the S2, P3, SG1, and SG4 fractions may reflect slightly higher expression levels of these proteins in synapsin double knockout mice; however, the specificity of the increases in specific subcellular fractions rather than generalized increases in all subcellular fractions makes this possibility less likely and implies a significant change in their subcellular distribution. Other proteins that bind nNOS, including PIN͞LC8͞DLC8 (14), heat shock protein 90 (40), and phosphofructokinase (41), may also function as targeting proteins for nNOS. Interestingly, a PIN͞LC8-binding consensus sequence has been found in dynamin (42), providing another mechanism by which nNOS may be targeted presynaptically.…”

Reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise

“…NOS enzymes have tetrahydrobiopterin as a cofactor that mediates coupling of oxygen reduction to haem catalysed L-arginine oxidation to form NO and citrulline. In recent years in has become apparent that when NOS enzymes lack substrate (arginine), cofactor (tetrahydrobiopterin) or specific protein-protein interactions (Xia et al 1996;Cosentino et al 1998;Bender et al 1999;Pou et al 1999;Song et al 2002;Ou et al 2003), the oxygen reduction and arginine oxidation become 'uncoupled' leading to generation of superoxide by the enzymes. In endothelial cells this uncoupling leads to superoxide-mediated endothelial dysfunction (Cosentino & Lü scher 1999;Cosentino et al 2001).…”

GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

“…Over the years, many cytosolic heme binding proteins have been described (11) but none have been shown to function in intracellular heme transport or heme insertion reactions. In fact, the studies to date have not identified any cytosolic heme transport proteins in mammals and only suggest that heme insertion may be a postendoplasmic reticulum process and may involve a chaperone (HSP90) (12)(13)(14)(15).…”