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...

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor⅐hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)⅐Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90⅐Hop⅐Hsp70⅐Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR⅐Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358 -7366). Hop has been considered essential for the formation of receptor⅐Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR⅐Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.The steroid receptors are recovered from cells as multiprotein heterocomplexes containing a dimer of Hsp90, substochiometric amounts of Hsp70, 1 an acidic 23-kDa protein, p23, and a tetratricopeptide repeat domain protein, such as immunophilin or protein phosphatase 5 (for review see Refs. 1 and 2). The steroid receptor⅐Hsp90 heterocomplexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with reticulocyte lysate (3, 4). Inasmuch as the glucocorticoid receptor (GR) 2 must be associated with Hsp90 for it to have steroid binding activity (5), incubation of Hsp90-free GR with reticulocyte lysate results in generation of steroid binding activity in direct proportion to the number of GR⅐Hsp90 heterocomplexes that are assembled (6). Hsp90 binds directly to the ligand-binding domain (LBD) of the GR (1), and complexing with Hsp90 also opens up both thiol moieties (7) and trypsin cleavage sites (8, 9) in the LBD to attack by a thiol-derivatizing agent and the protease. These biochemical observations, coupled with data derived from GR mutants (10), support the idea (6, 11) that the Hsp90 heterocomplex assembly system in reticulocyte lysate directs an ATP-dependent partial unfolding of the GR LBD, thus opening the hydrophobic steroid-binding cleft to access by steroid.The heterocomplex assembly system has been reconstituted (12-16), and five proteins, including Hsp90, Hsp70, Hop (60-kDa Hsp organizer protein), Hsp40, and p23, participate in the ATP/Mg 2ϩ -dependent and K ϩ -dependent assembly process (for review of heterocomplex assembly see Refs. 17 and 18). Besides Hsp90 itself, the only component of this Hsp90-based chaperone system proven to be essential for both GR⅐Hsp90 heterocomplex...

The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structurebased drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and wholeanimal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK I ϭ 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 g/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammationinduced anemia model in rats. JNJ-42041935 (100 mol/kg, once a day for 14 days) was effective in reversing inflammationinduced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-R (HIF1R). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand-and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

The 90-kD molecular chaperone hsp90 is the key component of a multiprotein chaperone complex that facilitates folding, stabilization, and functional modulation of a number of signaling proteins. The components of the animal chaperone complex include hsp90, hsp70, hsp40, Hop, and p23. The animal Hop functions to link hsp90 and hsp70, and it can also inhibit the ATPase activity of hsp90. We have demonstrated the presence of an hsp90 chaperone complex in plant cells, but not all components of the complex have been identified. Here, we report the isolation and characterization of soybean (Glycine max) GmHop-1, a soybean homolog of mammalian Hop. An analysis of soybean expressed sequence tags, combined with preexisting data in literature, suggested the presence of at least three related genes encoding Hop-like proteins in soybean. Transcripts corresponding to Hop-like proteins in soybean were detected under normal growth conditions, and their levels increased further in response to stress. A recombinant GmHop-1 bound hsp90 and its binding to hsp90 could be blocked by the tetratricopeptide repeat (TPR) domain of rat (Rattus norvegicus) protein phosphatase 5. Deletion of amino acids 325 to 395, adjacent to the TPR2A domain in GmHop-1, resulted in loss of hsp90 binding. In a minimal assembly system, GmHop-1 was able to stimulate mammalian steroid receptor folding. These data show that plant and animal Hop homologs are conserved in their general characteristics, and suggest that a Hop-like protein in plants is an important cochaperone of plant hsp90.The highly conserved and abundant molecular chaperone hsp90 is distinct from other chaperones in that it plays a key role in signal transduction networks, cell cycle control, protein degradation, and genomic silencing (Young et al., 2001). A critical dependence on hsp90 has been established for animal steroid hormone receptors (SRs), several Ser/Thr and Tyr kinases, and other distinct proteins (Pratt and Toft, 1997;Buchner, 1999). Two key features regarding the mechanism of hsp90 action have emerged over the last decade: (a) hsp90 lies at the center of a multiprotein chaperone complex that facilitates the folding of client proteins into their stable or activatable conformations, and (b) hsp90 is an ATPdependent chaperone. The study of hsp90 complexes, in particular those involving animal SRs, has revealed that five crucial chaperone components participate in the conformational regulation of hsp90 client proteins. These include hsp90 and hsp70 and their cochaperones Hop, p23, and hsp40. The high-M r immunophilins are also recovered in hsp90 complexes, but these appear to be nonessential in receptor folding assays (Pratt and Toft, 1997). The cochaperone Cdc37p/p50 cdc37 is predominantly found in hsp90-kinase complexes (Pratt and Toft, 1997).Hop (hsp70-and hsp90-organizing protein) derives its name from its role as an adapter protein that can bind to both hsp90 and hsp70 simultaneously, bringing them into close proximity (Chen and Smith, 1998). It is proposed that hsp70 first co...

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90⅐Hop⅐hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR⅐hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90⅐Hop⅐hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for ϳ30% of the hsp90 and ϳ9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR⅐hsp90 assembly activity resides in the peak containing Hopbound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90⅐hsp70 complexes with a stoichiometry of 2:1.Unliganded steroid receptors exist in cytosols in a heterocomplex with the ubiquitous protein chaperone hsp90 1 (for review, see Ref. 1). Hsp90 binds to the ligand binding domain (LBD) of the receptors (1), and the glucocorticoid receptor (GR) LBD must be bound to hsp90 for the receptor to have high affinity steroid binding activity (2, 3). The receptor⅐hsp90 heterocomplexes are assembled by a multiprotein chaperone system that was first studied in reticulocyte lysate (4, 5). Both biochemical data (6) and data from GR mutants (7, 8) support a model (3) in which the hydrophobic ligand binding cleft in the LBD is opened to access by steroid during heterocomplex assembly. The assembly system in reticulocyte lysate has been reconstituted (9), and a mixture of five purified proteins, hsp90, hsp70, 2 Hop, hsp40, and p23, is now used to achieve optimal receptor⅐hsp90 heterocomplex assembly (10, 11).The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR⅐hsp90 heterocomplex assembly (12). Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (13), and assembly proceeds faster when Hop is present to bring the two essential chaperones together (12). These complexes also contain small amounts of the hsp70 co-chaperone hsp40 (10), and together they form the hsp90/hsp70-based chaperone "machinery." The chaperone machinery can be prepared simply by mixing purified components, or it can be immunoadsorbed from reticulocyte lysate with a monoclonal antibody against Hop (10,14). When mixed with immunoadsorbed GR, the immunoadso...

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.