Hsp90 does not bind steroid receptors by itself. As determined largely in cell-free assembly assays with rabbit reticulocyte lysates, the assembly of the PR and GR with Hsp90 requires the participation of multiple chaperone components in an ordered assembly pathway. Three assembly stages for PR complexes have been described (Smith et al., 1995 ). In the early stage, Hsp70 binds free PR, perhaps with assistance from an Hsp40/DnaJ homolog. One of the Hsp70-binding proteins, Hip, also appears in early complexes. An intermediate complex is formed in which the receptor monomer is associated with Hsp70, the Hsp70-binding protein Hip, the Hsp70-/Hsp90-binding protein Hop, and Hsp90. In the final assembly stage, Hsp70, Hip, and Hop are absent from the PR complex, but Hsp90 remains with the Hsp90-associated protein p23 and one of the TPR-containing immunophilins.
Based on a combination of time course studies, chaperone-specific depletions, introduction of mutant chaperones, inhibitor studies, and reconstitution studies, it has been established that the ordered pathway described above is required for the assembly and maintenance of functional receptor complexes (Pratt and Toft, 1997). A key mechanistic element of this pathway involves the apparent roles of Hip and Hop as adaptors that help target Hsp90 to a preexisting Hsp70-receptor complex (Chen et al., 1996b; Prapapanich et al., 1998). Another key element is the p23-dependent stabilization of the interaction of Hsp90 with the receptor (Dittmar et al., 1996; Hutchison et al., 1995; Johnson and Toft, 1995) and the resulting establishment of a high affinity hormone-binding conformation (Scherrer et al., 1990; Smith, 1993). The functions of the Hsp90-associated immunophilins in mature receptor complexes have not yet been established. Furthermore, many of the details relating to transitions from one assembly stage to the next are vaguely defined, and there may be undiscovered components that participate in a highly transient or off-pathway manner. Nevertheless, the basic outline for the pathway of chaperone interactions resulting in functional steroid receptor complexes is well established.
Hormone binding to receptors results in their dissociation from Hsp90 and other chaperones, but hormones are not required to trigger dissociation of receptor complexes. In fact, chaperone interactions with receptors are highly transient at physiological temperatures (Smith, 1993; Smith et al., 1995). In the absence of bound hormone, however, dissociated receptor subunits quickly reassociate with Hsp70 and proceed through the assembly steps, generating a steady state assembly cycle. For the PR, at least, it appears that hormone binding blocks the binding of Hsp70 and re-entry of the PR into the assembly pathway.
Binding of Hsp90 and other chaperone components to steroid receptors is localized to the ligand binding domain (Carson-Jurica et al., 1989; Chambraud et al., 1990; Gehring and Arndt, 1985; Pratt et al., 1988; Schowalter et al., 1991), and chaperone interactions can clearly influence the conformation of this domain, as shown by chaperone-dependent acquisition and stabilization of high affinity hormone binding. But do chaperones complexed with steroid receptors function solely in folding of the ligand binding domain? Several observations argue against this. First, the estrogen receptor is assembled into complexes similar to those of the PR and GR, but the estrogen receptor does not require continued chaperone interactions for hormone binding. Second, steroid receptors lacking chaperones are competent for dimerization and deoxyribonucleic acid (DNA) binding in the absence of hormone. Hsp90 and other chaperone components in mature complexes mask the DNA binding domain of the receptor (Cadepond et al., 1991) and inhibit dimerization until chaperone interactions are interrupted, typically as a consequence of hormone binding. Third, dissociation of chaperones from receptors correlates with an increased rate of proteolytic degradation of the receptors in intact cells (Segnitz and Gehring, 1997; Whitesell and Cook, 1996). Thus, steroid receptors appear to be adapted for extended chaperone interactions that persist beyond basic folding steps and serve to regulate receptor function at various levels (for further discussions, see Nair et al., 1996; Smith, 1993; Smith et al., 1995). Enhancements of hormone binding and proteolytic half-lives can be considered activating functions, whereas inhibitions of receptor dimerization and DNA binding are repressive functions. Collectively, these activities maintain the receptor in a quiescent state that is competent for binding and responding to hormone.
As mentioned above, many other signaling proteins are targets for Hsp90 and may undergo interactions with multichaperone assemblies similar to those observed with steroid receptors (Nair et al., 1996, and references cited therein). However, several target-specific interactions that relate to Hsp90 partner proteins have been recognized. The three large immunophilins, i.e., FKBP52, FKBP51, and Cyp40, have each been recovered in individual steroid receptor complexes, but there is a clear preference for FKBP51 over the other immunophilins in PR and GR complexes assembled in vitro (Barent et al., 1998). The protein phosphatase PP5 is a TPR-containing protein that competes with immunophilins for Hsp90 binding and may associate preferentially with GR complexes (Silverstein et al., 1997). Another Hsp90-binding TPR protein appears in arylhydrocarbon/dioxin receptor complexes (Ma and Whitlock, 1997), and CDC37/pp50 appears preferentially together with Hsp90 in many kinase complexes (reviewed by Hunter and Poon, 1997), although chaperone interactions with the heme-regulated eIF2 kinase are more similar to those with steroid receptors (Matts et al., 1992; Uma et al., 1997, 1998; Xu et al., 1997). None of the Hsp90 accessory proteins has a clearly defined function in the respective signal protein complexes, but each may modulate the actions of Hsp90 or the target protein in a distinct manner.
Many heritable and acquired diseases result solely from the loss of wild-type protein activity, because of a mutation that disrupts a critical function of the gene product. On the other hand, some disease states result from production of a mutant protein that misfolds and acquires a novel activity (for example, a tendency to form aggregates) that has pathological consequences. Several human diseases involving protein misfolding have been cataloged in recent reviews (Ruddon et al., 1996; Thomas et al., 1995; Welch and Brown, 1996). A potential confounding factor in these diseases that has been largely overlooked is the possibility that different genetic backgrounds with distinctive patterns of chaperone expression and function might enhance or repress the phenotype of misfolding mutants. Furthermore, mutation of a chaperone component could possibly underlie certain disease states, with the phenotype depending on the range and sensitivity of particular protein substrates for the mutant chaperone. Here we address a few representative disease states, discuss how protein misfolding and chaperones participate in the disease process, and then consider potential treatments that target chaperone activity.
Chaperones are highly sophisticated protein machines that assist the folding of RNA molecules or other proteins. Their function is generally thought to require a fine-tuned and highly conserved structure: despite the recent recognition of the widespread occurrence of structural disorder in the proteome, this structural trait has never been generally considered in molecular chaperones. In this review we give evidence for the prevalence of functional regions without a well-defined 3-D structure in RNA and protein chaperones. By considering a variety of individual examples, we suggest that the structurally disordered chaperone regions either function as molecular recognition elements that act as solubilizers or locally loosen the structure of the kinetically trapped folding intermediate via transient binding to facilitate its conformational search. The importance of structural disorder is underlined by a predictor of natural disordered regions, which shows an extremely high proportion of such regions, unparalleled in any other protein class, within RNA chaperones: 54.2% of their residues fall into disordered regions and 40% fall within disordered regions longer than 30 consecutive residues. Structural disorder also prevails in protein chaperones, for which frequency values are 36.7% and 15%, respectively. In keeping with these and other details, a novel "entropy transfer" model is presented to account for the mechanistic role of structural disorder in chaperone function.—Tompa, P., Csermely, P. The role of structural disorder in the function of RNA and protein chaperones.
Key Words: chaperone evolution • chaperone function • entropy transfer • intrinsically unstructured protein