A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.

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 M 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 hemebinding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOSdependent 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 M, 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.The endothelial and neuronal isoforms of nitric-oxide synthase (NOS) 1 have been reported to exist in heterocomplexes with hsp90 (1, 2). These proteins join a list of numerous other signaling proteins, including steroid receptors, some transcription factors, and a variety of protein kinases, that are associated with and regulated by hsp90 (for a review, see Ref.3). These signaling protein⅐hsp90 heterocomplexes are assembled in an ATP-dependent process by a five-protein system in which hsp90 and hsp70 are essential assembly components and Hop, hsp40, and p23 function as non-essential co-chaperones (4). One of the most studied hsp90-bound proteins is the glucocorticoid receptor (GR), which must be associated with hsp90 to have steroid binding activity (5, 6). Hsp90 binds directly to the ligand-binding domain of the GR (3), and biochemical data (7) coupled with data from GR mutants (8, 9) support the notion (6) that formation of a complex with hsp90 opens up a hydrophobic pocket in the ligand-binding domain to access by steroid. We have proposed a similar model for neuronal NOS (nNOS) in which the hsp90-based chaperone machinery acts in vivo to open the heme-binding cleft in heme-deficient apo-nNOS to access by heme (2).In contrast to the observations with steroid receptors and nNOS, it has been proposed that hsp90 regulates endothelial NOS (eNOS) through an allosteric mechanism. GarciaCardeñ a et al.(1) were able to demonstrate direct activation of purified eNOS catalytic activity by purified hsp90 in the absence of ATP, hsp70, and th...

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS and the inhibition of heat shock protein 90 (hsp90)-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin-conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient nNOS (apo-nNOS) is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of aponNOS and enhance its degradation in vivo. In the current study, we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with N G -nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.

Nitric oxide synthase (NOS) is a highly regulated enzyme that produces nitric oxide, a critical messenger in many physiological processes. In this perspective, we explore the role of proteolytic degradation of NOS, in particular the inducible and neuronal isoforms of NOS, as a mechanism of regulation of the enzyme. The ubiquitin-proteasome and calpain pathways are the major proteolytic systems identified to date that are responsible for this regulated degradation. The degradation of NOS is affected by diverse agents, including glucocorticoids, caveolin, neurotoxic compounds, and certain NOS inhibitors. Some irreversible inactivators of NOS enhance the proteolytic degradation of the enzyme, and this property may be of great importance in understanding the biological effects of these inhibitors, some of which are being developed for clinical use. Analogies with the regulated degradation of liver microsomal cytochromes P450, which are related to NOS, provide a framework for understanding these processes. Finally, a new perspective on the regulation of NOS by hsp90-based chaperones is presented that involves facilitated heme insertion into the enzyme.Nitric oxide, the radical metabolite formed from the metabolism of L-arginine by nitric oxide synthase (NOS), has been shown to be 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 (Moncada et al., 1991;Burnett et al., 1992;Forstermann et al., 1994;Schmidt and Walter, 1994). There have been several reviews, one of them very recent (Alderton et al., 2001), on the structure, function, and inhibition of the three major isoforms of NOS: the neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Although various aspects of the regulation of these enzymes have also been reviewed, the focus of the current perspective on the regulated proteolytic degradation of NOS has not. Since certain NOS inhibitors selectively enhance the degradation of NOS protein, this regulatory process is likely to be of importance in understanding the biological actions of NOS inhibitors, some of which are currently being tested for human use. We also present a perspective on the role of hsp90-based chaperones in affecting the proteolysis of NOS by regulating heme insertion and formation of the active dimeric form. Proteolysis of NOSSelective proteolytic degradation of NOS is a mechanism for regulation of the enzyme. For example, transforming growth factor-␤ enhances the degradation of iNOS in interferon-␥-treated mouse peritoneal macrophages and causes suppression of NO release from these cells (Vodovotz et al., 1993). The glucocorticoid-mediated suppression of iNOS expression in IL-1␤-treated rat glomerular mesangial cells (Kunz et al., 1996) and interferon-␥-treated murine macrophage cell line RAW 264.7 (Walker et al., 1997) is due, in part, to increas...

Purpose: To evaluate rheological properties, in vitro dissolution, and in vivo ocular pharmacokinetics of loteprednol etabonate (LE) (submicron) ophthalmic gel 0.38%. Methods: The viscosity of the LE gel 0.38% formulation was measured with a controlled stress rheometer. Dissolution kinetics were evaluated in a fixed-volume and flow-through assay. Rabbits received a single instillation of LE (submicron) gel 0.38% (both eyes), and concentrations of LE in ocular tissues were determined through 24 h by liquid chromatography with tandem mass spectrometry. Where indicated, comparators included micronized LE gel 0.38%, 0.5% (Lotemax ® gel), and 0.75%. Results: LE (submicron) gel 0.38% exhibited shear-thinning characteristics similar to LE gel 0.5% with nearly identical yield stress. LE (submicron) gel 0.38% released 2.6-fold more LE into the dissolution medium than micronized LE gel 0.5% over 30 s in the fixed-volume dissolution assay, and submicron LE attained higher concentrations of dissolved LE than micronized LE gel 0.38% in the flow-through dissolution assay. In rabbits, the maximal concentration and area-under-the-curve over 24 h for LE in aqueous humor were 2.5- and 1.8-fold higher, respectively, for LE (submicron) gel 0.38% versus micronized LE gel 0.5% (both P < 0.001). Pharmacokinetic parameters were similar for most other tissues. Conclusions: LE (submicron) gel 0.38% demonstrated similar rheological properties to micronized LE gel 0.5% but faster dissolution, thus providing similar or higher LE concentrations in the aqueous humor, cornea, and iris-ciliary body after ocular dosing in rabbits despite a lowered concentration of drug in the formulation.