Kimon C. Kanelakis

Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein

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 receptor·hsp90 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.

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

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 GR·hsp90 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 nNOS·hsp90 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 nNOS·hsp90 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.

The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)·Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90·Hop·Hsp70·Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR·Hsp90 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 receptor·Hsp90 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 GR·Hsp90 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.

Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR)·hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR·hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of ∼0.03, and its elimination by immunoadsorption did not affect GR folding and GR·hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR·hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR·hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.

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 μm 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 μm, 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.

Characterization of a Plant Homolog of Hop, a Cochaperone of Hsp90

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.

Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes.

hsp90 and hsp70 are essential components of a five-protein system, including also the nonessential cochaperones Hop, hsp40, and p23, that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding pocket to access by steroid. The first event in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR, which primes the receptor for subsequent ATP-dependent activation by hsp90 [Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., and Pratt, W. B. (2000) J. Biol. Chem. 275, 18054-18060]. Here, we demonstrate that, during the priming step, ATP-bound hsp70 is converted to GR-bound hsp70 that is approximately 1/3 in the ADP- and approximately 2/3 in the ATP-dependent conformation. In the second step, hsp90, which is provided in the non-nucleotide-bound state, is converted to GR-bound hsp90 in the ATP-dependent conformation. The ATPase activity of hsp70 is K(+)-dependent, and the priming step is K(+)-dependent. Surprisingly, the subsequent hsp90-dependent step, which is rate-limiting for receptor activation, is also potassium-dependent. This suggests that GR-bound hsp70 is also converted from the ATP-dependent to the ADP-dependent conformation while it cooperates with hsp90 to activate steroid binding activity. Because the priming step requires both sustained high levels of ATP and YDJ-1 for optimal activity and because both steps require potassium, we predict that receptor-bound hsp70 undergoes iterative ratcheting between its ATP- and ADP-dependent conformations in opening the hydrophobic steroid binding pocket.

Regulation of glucocorticoid receptor ligand-binding activity by the hsp90/hsp70-based chaperone machinery.

Publisher Summary This chapter focuses on the regulation of glucocorticoid receptor ligand-binding activity by the hsp90/hsp70-based charperone machinery. Nearly 100 proteins are known to be regulated by hsp90. Most of these substrates, or ‘‘client’’ proteins, are involved in signal transduction, and they are assembled into heterocomplexes with hsp90 by a multiprotein hsp90/hsp70-based chaperone machinery. Hop (Hsp organizing protein) is a protein with two tetratricopeptide repeat (TPR) domains that bind independently to hsp90 and hsp70 to bring the two chaperones together in an hsp90-Hop-hsp70-hsp40 machinery that is labeled as a “foldosome.” The purified five-protein system has allowed to define essential versus nonessential chaperones for opening the steroid-binding cleft and for ‘‘stable’’ GR-hsp90 heterocomplex assembly. The definition of a purified five-protein system that assembles heterocomplexes between nuclear receptors, and hsp90 is enabling advances in the understanding of the mechanism by which the hsp90/hsp70-based chaperone machinery functions. It is likely that the five-protein system described in the chapter will regulate the function of a wide variety of proteins. The five-protein system has also been used to reconstitute a functional hepadenovirus reverse transcriptase, indicating that the system may be responsible for hsp90 regulation of a variety of other biological processes that are dependent upon hsp90.

Correlation between pregnanesteroid conformation, receptor affinity, and anti-natriuretic effect

The aim of this study was to correlate mineralocorticoid action and steroid structure. Inasmuch as Na(+) retention follows a parabolic dose-response curve for most pregnanesteroids, the second-order coefficient of the function was used as a representative factor for this bipartite biological effect. The C(3)=O/D angle of the ligands was correlated with both Na(+)-retaining activity and binding affinity for the mineralocorticoid receptor. Because some steroids exhibit identical functional groups and different conformational structure, we also postulate that the flat conformation of a pregnanesteroid determines its Na(+)-retaining capacity in vivo. No correlations were found in vitro, which demonstrates the multifactorial nature of the second-order coefficient determined in vivo under more complex and interactive conditions that include various pre-receptor variables. These findings may allow the estimation of the putative biological activity of a given steroid simply by knowing its conformational structure, which may be important for designing compounds in a pharmaceutical setting.

Regulation of G protein signaling by the 70kDa heat shock protein

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha(2A) adrenergic receptor (α(2A)-R) by the ubiquitous stress-inducible 70kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α(2A)-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α(2A)-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α(2A)-R-catalyzed [(35)S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine(1A) receptor (5-HT(1A)-R). In heat-stressed CHO cells, the α(2A)-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α(2A)-R compared to the 5-HT(1A)-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.