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...

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor⅐hsp90 heterocomplexes. We have reconstituted a minimal receptor⅐hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 In this work, we show that addition of p23 to native GR⅐hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR⅐hsp90 heterocomplexes prepared with the minimal (hsp90⅐p60⅐hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR⅐hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90⅐p60⅐hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR⅐hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90⅐p60⅐hsp70 forms a GR⅐hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR⅐hsp90 heterocomplex to inactivation and disassembly.

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of 125 I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The hemedeficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of nonfunctional enzyme.

Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide (1-4). Nitric oxide is a signaling molecule that is involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (5-8). It has been noted (9) that because nitric oxide is not stored, released, or inactivated after synaptic release by conventional regulatory mechanisms the biosynthetic regulation of the enzyme is of great importance. For the neuronal isoform the Ca 2ϩ -mediated activation is of prime importance. However, the factors that regulate proteolytic degradation of the enzyme have not been investigated. One approach that has been successfully utilized for the study of the proteolytic turnover of other P450 cytochromes is the use of suicide inactivators (10 -13). We wished to utilize this approach for the study of NOS turnover.Suicide inactivators are chemically inert molecules that mimic the natural substrate of the enzyme and become metabolized to a highly reactive intermediate that can covalently alter important active site entities, resulting in inactivation of the enzyme (14). In effect, the compound causes the enzyme to catalyze its own demise. Because the compound must not only have affinity for the active site but also must be metabolized in a manner to generate a reactive intermediate that is in close proximity to an important active site entity, these agents have the potential to be highly specific in vivo. In addition, they react with the form of the enzyme that is engaged in catalysis and thus are useful mechanistic probes into the nature of the bioactivation reaction. In some cases, these agents covalently alter P450 cytochromes in a manner that enhances their proteolysis and turnover (10 -13, 15-17).Guanabenz, a clinically used antihypertensive agent with a guanidino moiety, was recently shown, with the use of brain and penile cytosol, to be a metabolism-based inactivator of neuronal nitric-oxide synthase (nNOS) (18). Moreover, the treatment of rats with guanabenz was found to cause not only a decrease in activity, but also a concomitant loss of immunodetectable nNOS protein. To further understand the molecular mechanisms responsible for the loss of nNOS protein in vivo, we chose to model the effects of guanabenz with the use of HEK 293 cells stably transfected with nNOS. In the current study, we have shown that guanabenz causes the enhanced proteolytic turnover of nNOS. Suicide inactivators of nNOS, such as N G -methyl-L-arginine or N 5 -(1-iminoethyl)-L-ornithine, also caused the enhanced proteolytic degradation of the enzyme, whereas the slowly reversible inhibitor N G -nitro-L-arginine or the reversible inhibitor 7-NI did not. Studies with protease inhibitors indicate that the proteasome is responsible, in part, for r...

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It is established that agmatine, an endogenously formed decarboxylated arginine, is a weak competitive inhibitor of neuronal nitric-oxide synthase (nNOS) with an apparent K i value of 660 M [Biochem J 316:247-249, 1996]. Although agmatine is known to bind to ␣-adrenergic and imidazoline receptors, it has been suggested that some of the pharmacological actions of agmatine, such as the prevention of morphine tolerance, may be due to the inhibition of nNOS. In the current study, we have discovered that agmatine, at concentrations much lower than the reported K i value, leads to a time-, concentration-, NADPH-, and calmodulin-dependent irreversible inactivation of nNOS. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (K i ) and a pseudo first-order inactivation constant (k inact ) of 29 M and 0.01 min Ϫ1 , respectively. As determined by high-performance liquid chromatography analysis, the mechanism of inactivation involves alteration of the prosthetic heme moiety of nNOS, in part to protein-bound products. Moreover, we discovered that agmatine causes a 3-fold increase in the NADPH oxidase activity of nNOS leading to the production of H 2 O 2 and is a likely cause for the inactivation of the enzyme. Both the inactivation of nNOS and the oxidative stress produced should now be considered in the pharmacological actions of agmatine as well as provide insight into the potential biological effects of endogenously formed agmatine.

It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [ 14 C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.

Cellular stress or injury can result in mitochondrial dysfunction, which has been linked to many chronic neurological disorders including amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). Stressed and dysfunctional mitochondria exhibit an increase in large conductance mitochondrial membrane currents and a decrease in bioenergetic efficiency. Inefficient energy production puts cells, and particularly neurons, at risk of death when energy demands exceed cellular energy production. Here we show that the candidate ALS drug dexpramipexole (DEX; KNS-760704; ((6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) and cyclosporine A (CSA) inhibited increases in ion conductance in whole rat brain-derived mitochondria induced by calcium or treatment with a proteasome inhibitor, although only CSA inhibited calcium-induced permeability transition in liver-derived mitochondria. In several cell lines, including cortical neurons in culture, DEX significantly decreased oxygen consumption while maintaining or increasing production of adenosine triphosphate (ATP). DEX also normalized the metabolic profile of injured cells and was protective against the cytotoxic effects of proteasome inhibition. These data indicate that DEX increases the efficiency of oxidative phosphorylation, possibly by inhibition of a CSA-sensitive mitochondrial conductance.