I. Protein Folding in the Cell

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Successful expression of polypeptide gene products requires transcription machinery to generate messenger ribonucleic acids and translation machinery to synthesize polypeptide chains but the protein product is functional, and thus the gene is effectively expressed, only when the polypeptide chain is folded into its native three-dimensional conformation, appropriately localized within or secreted from the cell, and, in many instances, properly assembled into multicomponent complexes. Although the amino acid sequence alone is sufficient to dictate the native conformation of small proteins in vitro (Anfinsen, 1973), most polypeptides would fail to fold efficiently in the highly concentrated, complex, cellular environment without the assistance of yet another type of machinery. This latter type of machinery involves the so-called molecular chaperones, i.e., proteins adapted to facilitate protein folding. The chaperone concept was first proposed by Ellis (1987), and a large, highly active field of research has since developed to investigate the functions and physiological implications of molecular chaperones. Major aspects of this field have been recently reviewed (Buchner, 1996; Hartl, 1996), and many details have been compiled in a more comprehensive manner (Gething, 1997).

There is an inherent problem in the folding of nascent polypeptide chains. Whether synthesized on free ribosomes in the cytoplasm or on ribosomes associated with endoplasmic reticulum (ER),^b nascent protein chains emerge in a linear manner. Consequently, hydrophobic stretches and other potentially interactive sites must sometimes wait for downstream sequences to emerge before appropriate folding interactions can be established. Inappropriate interactions can readily lead to poorly reversible conformations and aggregations that reduce the efficiency of native folding reactions (Jaenicke, 1995). Molecular chaperones reversibly interact with nascent chains to minimize off-pathway interactions and increase the yield of native folded protein.

In addition to the folding of nascent polypeptide chains, other cellular processes involve protein folding that requires chaperone participation. One of these is the transport of previously synthesized proteins across cell membranes, as best understood for mitochondrial protein import (Langer et al., 1997). Many mitochondrial proteins are encoded by nuclear genes, synthesized on free ribosomes in the cytoplasm, and subsequently transported across one or both mitochondrial membranes. Proteins are translocated across the mitochondrial membranes in a linear manner as unfolded chains, and there are both cytoplasmic and mitochondrial chaperones that participate in the unfolding and refolding pathways.

	II. Molecular and Chemical Chaperones

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Several physical and chemical conditions favor inappropriate folding of proteins and are thus hazardous to cells. Among these proteotoxic conditions are elevated temperature, anoxia, and exposure to ethanol, heavy metals, or other chemical denaturants. It has been recognized for >30 years that mild temperature elevation can induce a so-called heat shock response in all cells (reviewed by Lindquist and Craig, 1988). This response is characterized by a rapid shutdown of the synthesis of most proteins, with a dramatic transient increase in the synthesis of a small set of proteins, accordingly called heat shock proteins (Hsps). A similar response is observed after other proteotoxic insults, so the more general terms stress response and stress proteins are sometimes used. For years the function of Hsps was unknown, but studies in the past 10 years have defined the major Hsps as central components of the molecular chaperone/protein folding machinery. Thus, stress responses are evolutionary adaptations to quickly resolve misfolded proteins and restore the normal protein folding environment of cells. In addition, an initial sublethal exposure to heat or other stresses can condition cells for enhanced survival during exposure to subsequent, even more severe, stresses (Gerner and Schneider, 1975); this is commonly termed thermotolerance and is largely attributable to induction of Hsps (reviewed by Parsell et al., 1993).

Many Hsps and their associated co-chaperones are constitutively expressed in all cells. There are several multigene Hsp families, and individual genes within families differ to varying degrees with respect to sequence and expression patterns, as well as the function and subcellular localization of the respective gene products (table 1). Major Hsp families, named to reflect the approximate molecular size (in kilodaltons) of family members, are Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and the small Hsp family (typically 20 to 25 kDa). The exact function or range of functions for each of the families and its individual members is being actively investigated in many laboratories. Hsp70, Hsp40, and Hsp60 family members play important roles in nascent chain folding (Hartl, 1996). Hsp70 members are also major components in membrane translocation processes. The small Hsps have important functions in disaggregation or degradation of misfolded complexes (Gething, 1997), but it is not clear how important they are for nascent chain folding. Hsp90 also may have a role in nascent chain folding, but it is most notable for its numerous associations with important regulatory proteins (Pratt and Toft, 1997). Hsp100 family members, such as Hsp104, play an important role in thermotolerance in yeast (Lindquist and Kim, 1996; Sanchez and Lindquist, 1990; Schirmer et al., 1996). An overall increase in Hsp levels correlates with the acquisition of thermotolerance, but increased Hsp104 levels appear to be the critical factor. Importantly, Hsp104 is adept at preventing and reversing protein aggregation (Parsell et al., 1994). In addition to these major Hsp families, there are many additional proteins whose expression is responsive to various stresses, but these are not discussed except as they pertain to the topics presented here.

Although much remains to be learned in this relatively new field, some chaperone-mediated processes have been characterized in detail. Illustrative of the coordination among chaperones is the typical pathway for nascent chain folding in Escherichia coli (reviewed by Martin and Hartl, 1997). This pathway involves Hsp40, Hsp70, Hsp60, and Hsp10 family members. After approximately 50 residues on the nascent chain emerge from the ribosome, DnaJ (an Hsp40) and DnaK (an Hsp70) have access to the growing polypeptide. As synthesis is completed, the nascent chain, if no more than approximately 50 kDa, is passed from DnaJ/DnaK to the lumen of a double-ring complex formed by 14 subunits of GroEL (an Hsp60). The GroEL ring can be capped by a seven-subunit ring of GroES (an Hsp10), transiently enclosing the nascent polypeptide within the GroEL cavity. The nascent chain undergoes rounds of binding and release from GroEL/GroES complexes until its folding is complete. DnaK and GroEL interactions with the incompletely folded substrate are adenosine triphosphate (ATP)-dependent and regulated by ATP hydrolysis. In addition, at least one other protein, termed GrpE, participates as a partner protein of DnaK.

In eukaryotes, a typical pathway of chaperone interactions with cytoplasmic nascent chains has been more difficult to define, although Hsp40 and Hsp70 homologs are important components (Eggers et al., 1997; Frydman et al., 1994; Hansen et al., 1994). Netzer and Hartl (1997) recently pointed out that nascent chain folding in eukaryotes occurs more at the co-translational level, compared with posttranslational folding in prokaryotes, and co-translational folding correlates with the more common occurrence of multidomain proteins in eukaryotes. These differences may contribute to the dissimilarities of chaperone systems involved in nascent chain folding in prokaryotes versus eukaryotes.

Whereas GroEL is a general nascent chain factor in prokaryotes, the closest Hsp60 homologs in eukaryotes are in mitochondria and chloroplasts (essentially prokaryotic organelles). In the cytoplasmic compartment there is a family of proteins, termed T-complex polypeptides (TCPs), that are loosely homologous with GroEL and form double-ring complexes similar to that of GroEL (Kubota et al., 1995). TCP ring complexes are less abundant in cytoplasm than would appear to be necessary for general protein folding, so they may have a limited range of substrates. Significantly, tubulin and actin subunits are specific substrates that require the chaperoning of TCP ring complexes (Sternlicht et al., 1993; Yaffe et al., 1992).

The bacterial Hsp90 homolog Htpg is expressed at low levels and is nonessential (Bardwell and Craig, 1988). In marked contrast to prokaryotic systems, Hsp90 is typically the most abundant chaperone in eukaryotic cytoplasm, and its expression is essential (Borkovich et al., 1989). The potential role of Hsp90 in nascent chain folding is unresolved (Eggers et al., 1997). In in vitro refolding assays in a minimal buffer, eukaryotic Hsp90 is capable of holding heat-denatured enzymes in a competent state for refolding (Wiech et al., 1992), and Hsp90 has two apparent substrate binding sites (Scheibel et al., 1998; Young et al., 1997). However, based on a study in Saccharomyces cerevisiae that examined the chaperone function of Hsp90 in vivo (Nathan et al., 1997), Hsp90 probably is not required for nascent chain folding of most proteins but is required for a subset of proteins. In addition to nascent chain folding and refolding of denatured proteins, the regulatory interactions of Hsp90 with a variety of important signal transduction proteins have been extensively characterized.

There are several eukaryotic chaperone components that have no prokaryotic homologs. Some of these have activities similar to those of Hsp90, as determined in vitro in refolding assays (Freeman et al., 1996), perhaps generating redundancy in this potentially important cellular function. On the other hand, a strong case has been developed for their functioning as partner proteins or co-chaperones that directly interact with Hsp70 and/or Hsp90. Included in a growing list of Hsp70-associated partners are Hip/p48 (Hoehfeld et al., 1995; Prapapanich et al., 1996), Hop/p60 (Honore et al., 1992; Smith et al., 1993), Bag-1 (Hoehfeld and Jentsch, 1997; Takayama et al., 1995, 1997), RAP46 (Gebauer et al., 1997; Zeiner et al., 1997; Zeiner and Gehring, 1995), and p16, a member of the Nm23/nucleotide diphosphate kinase family (Leung and Hightower, 1997). These can have either positive or negative effects on Hsp70 activity, in some cases depending on the assay conditions. There are even more Hsp90-binding proteins. Most of the known Hsp90 partners contain a tetratricopeptide repeat (TPR) domain that is required for Hsp90 binding. One of these is the Hsp70-binding protein Hop, which can bind concomitantly with Hsp90 (Chen et al., 1996b; Lassle et al., 1997). Other Hsp90-associated TPR proteins belong to the immunophilin families of FK506-binding proteins (FKBPs) and cyclosporin-binding proteins, i.e., cyclophilins (Cyps). FKBP52/FKBP59/Hsp56, FKBP51, and Cyp40, each of which is expressed in many cell types, compete for binding to Hsp90 and have been noted in a variety of Hsp90 complexes (Nair et al., 1997, and references cited therein; Owens-Grillo et al., 1995; Radanyi et al., 1994; Ratajczak and Carrello, 1996). As with other immunophilin family members, the Hsp90-associated immunophilins have peptidylprolyl isomerase (PPIase) activity, but the importance of this activity is unknown (Barent et al., 1998). An additional TPR-containing, Hsp90-binding protein is the protein phosphatase PP5 (Chen et al., 1996a; Chinkers, 1994; Silverstein et al., 1997). Lacking any TPR motif is p23, an Hsp90 partner that stabilizes Hsp90 binding to various target proteins (Hutchison et al., 1995; Johnson et al., 1994; Johnson and Toft, 1995). A pathway of interactions involving Hsp70, Hsp90, and many of their partner proteins is presented in Section III.

The ER contains its own complex chaperone machinery (reviewed by Hammond and Helenius, 1995; Helenius et al., 1992; Ruddon and Bedows, 1997). Two of the major ER chaperones are Bip/Grp78 and Grp94/gp96, members of the Hsp70 and Hsp90 families, respectively. There are also unique chaperone components in the ER chaperone machinery, two of which are the Ca²⁺-binding proteins calnexin and calreticulin.

Compared with cytoplasm, the lumen of the ER is a distinct folding environment in which the redox potential is oxidizing, and there is a relatively high concentration of Ca²⁺. Other special aspects of protein synthesis and folding in the ER include the common glycosylation of emerging nascent chains, the frequent occurrence of disulfide bonds that stabilize polypeptide conformation and oligomerization, and the dual folding environment for proteins with transmembrane domains. A final consideration is that the ER is only a temporary location for most proteins, because they are usually destined for transit to the Golgi or beyond.

Nascent chains emerging into the ER lumen often are rapidly glycosylated on asparagine and glutamine side chains. As the nascent chain elongates, it associates with ER chaperones, in particular calnexin or calreticulin (which bind in a glycosylation-dependent manner) and/or Bip, but additional ER chaperones have also been detected in nascent chain complexes; this may reflect multichaperone complexes in the ER (Tatu and Helenius, 1997). After chain elongation is complete, the incompletely folded nascent chain may undergo sequential rounds of chaperone binding. Only fully folded chains and properly assembled oligomeric complexes exit efficiently from the ER and progress to the Golgi. Misfolded or unassembled proteins are retained in the ER by continued chaperone interactions, but the exact nature and combination of chaperone interactions vary with different substrates (Zhang et al., 1997b, and references cited therein). Interestingly, the eventual proteolytic degradation of retained proteins (whether integral membrane or soluble proteins) often occurs on cytoplasmic proteasomes through a poorly defined export mechanism (Kopito, 1997). The ER chaperone machinery has often been characterized as a quality control station, and, as seen in Section IV., this quality control function is relevant to several clinical conditions.

In addition to the protein components of the chaperone machinery, several low molecular weight osmolytes in cells are known to favor protein folding and stability (reviewed by Burg, 1995; Welch and Brown, 1996; Yancey et al., 1982). Glycerol and trehalose are among the more important protein-stabilizing compounds, but other carbohydrates, amino acids, and methylamines can also contribute. Not coincidentally, glycerol and trehalose are commonly recognized as general stabilizing agents for proteins in solution (reviewed by Schein, 1990). Intracellular concentrations of osmolytes can reach dramatically high levels after hypo-osmotic shock (Burg, 1995); moreover, a similar increase in the levels of some osmolytes has been noted after heat shock (Hottiger et al., 1987). To reflect their potentially important role in maintaining a cellular environment conducive for protein folding, these compounds have been termed chemical chaperones (Welch and Brown, 1996). The general mechanism for stabilization of protein structures by chemical chaperones probably relates to their ability to stabilize the hydration shell around proteins (Schein, 1990).

	III. Chaperone-Mediated Regulation of Signal Transduction Pathways

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Apart from their role in protein-folding processes, several components of the chaperone machinery appear to function as regulatory factors for a variety of signaling proteins. Hsp90 has been found to interact with multiple regulatory proteins, including the steroid hormone receptors, several transcription factors unrelated to steroid receptors, and various tyrosine and serine/threonine kinases (reviewed by Pratt and Toft, 1997). The progesterone receptor (PR) and glucocorticoid receptor (GR) are the most well characterized targets for Hsp90 regulation, and the following discussion centers on what has been learned from studying chaperone-steroid receptor interactions. Many of the chaperone components and mechanisms of interaction described for steroid receptors appear to pertain to kinases and other Hsp90 targets (outlined in fig. 1); however, there are some important target-specific differences.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chaperone interactions that regulate the activity of target proteins. Steroid receptors undergo an ordered series of chaperone interactions that regulate receptor activity in various ways (see text for details). Other target proteins may interact in a chaperone pathway similar to that for steroid receptors, or there may be alternate chaperone pathways that are target-specific. The assembly pathway has been described in detail only for steroid receptors. However, a common feature in most target pathways is the eventual association with Hsp90 and one or more Hsp90-binding partner proteins. Imph., immunophilin.

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.

	IV. Protein Misfolding in Disease

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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.

The fact that chaperone interactions play an important role in regulating key components of cellular growth and differentiation pathways suggests that they may be involved in the initiation and progression of human cancers. Dysfunction within the chaperone machinery resulting from mutation or altered expression of specific Hsps may give rise to neoplastic transformation in a unique way by impairing the activity, localization, or stability of multiple signal transduction molecules and/or transcription factors simultaneously. Consistent with this hypothesis, the cytoplasmic sequestration and aggregation of wild-type forms of the tumor suppressor protein p53 have been documented in neuroblastoma (Ostermeyer et al., 1996) and some breast cancers (Moll et al., 1992). A specific defect in chaperone function, however, has yet to be demonstrated in these tumors. The overexpression of certain Hsps in breast (Ciocca et al., 1993) and ovarian (Kimura et al., 1993) cancers has been reported to be associated with poor clinical prognosis, but the reason remains obscure. Given the general role of Hsps in cytoprotection, perhaps overexpression renders cells relatively resistant to conventional chemotherapeutic agents, but this possibility remains to be proven. Finally, our rapidly evolving understanding of the molecular mechanisms of oncogenesis suggests that chaperone interactions may actually provide a useful target for anticancer drug action.

Many of the common mutations in both tumor suppressor genes and dominantly acting oncogenes result in the expression of defective proteins that display unusually stable physical association with normal molecular chaperones. For example, soon after the transforming factor of the Rous sarcoma virus was identified as the constitutively activated tyrosine kinase v-Src, it was noticed that the kinase co-precipitated with several endogenous cellular proteins, including Hsp90 and Hsp70 (Hutchison et al., 1992; Oppermann et al., 1981). Moreover, through mechanisms that remain to be defined, complex formation appears to be required for transforming activity, because drugs that interfere with Hsp90 function revert v-Src-mediated transformation, despite persistently elevated intracellular tyrosine kinase activity (Kwon et al., 1992; Whitesell et al., 1994).

The interaction of Hsp90 and other chaperones with oncogenically mutated kinases appears to differ from that of the normal cellular counterparts primarily in the relative stability of chaperone associations with mutant kinases. As best demonstrated by Matts and colleagues (Hartson et al., 1996, 1998; Hartson and Matts, 1994), c-Src and the cellular Src-related kinase Lck also appear to depend on chaperone interactions for their activity.

Perhaps the most intriguing example of chaperone involvement in malignant transformation can be found in the rapidly evolving description of p53-mediated oncogenesis. Mutations of the p53 tumor suppressor gene are the most common molecular genetic defects found in human cancers (Harris and Hollstein, 1993). Most p53 mutations result in the expression of a protein of altered conformation that has lost its cell cycle checkpoint activity. Normal, wild-type p53 is a short-lived protein that is rapidly turned over via selective proteolysis in the ubiquitin-proteasome pathway (Maki et al., 1996). Presumably because of their aberrant conformations, however, many p53 mutants are retained within the chaperone machinery (Davidoff et al., 1992; Selkirk et al., 1994; Sepehrnia et al., 1996) and protected from ubiquitin conjugation and subsequent degradation. As a result, elevated levels of dysfunctional protein accumulate within the tumor cell. A simplified cartoon representation of the mechanisms by which altered chaperone interactions may contribute to p53-mediated oncogenesis is presented in fig. 2. Of note, mutant p53 proteins bound to Hsps no longer function as tumor suppressors, and some mutants may actually interfere with the function of normal p53 (which continues to be expressed from the remaining wild-type allele) by forming heterodimers with it (dominant negative effect). In addition, evidence exists that some mutant p53 proteins can directly activate inappropriate gene expression, contributing to oncogenesis (positive tumor-promoting effect) (Park et al., 1994). Recent work has demonstrated that benzoquinone ansamycin drugs can disrupt the extended chaperone interactions observed with mutant p53 and selectively destabilize mutant proteins without effecting the cellular levels of wild-type protein (Blagosklonny et al., 1995; Whitesell et al., 1997). Drug treatment, however, does not appear to restore wild-type transactivating activity to the mutant protein (Whitesell et al., 1998). Although drug treatment does not appear to restore tumor suppressor activity to mutant p53 protein, it may abrogate the positive transforming activities of mutant p53 within tumor cells. Moreover, in cells that are heterozygous for p53 mutation, destabilization of mutant p53 might restore function to protein expressed from the wild-type allele (Blagosklonny et al., 1995). It remains to be seen whether these interesting cell biological considerations can be translated into clinically useful chemotherapeutic strategies.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Simplified cartoon representation of mechanisms by which altered chaperone interactions may contribute to the transforming activity of oncoproteins such as mutant p53. A mutation in the primary amino acid sequence of the target protein leads to an altered conformation (steps 1 and 2). This mutant conformation is recognized by the chaperone machinery as "misfolded," resulting in extended interactions with chaperone complexes (step 3). As a result, the mutant protein is mislocalized within the cell and does not function properly (step 4). In addition, normal proteolytic degradation is impaired, and the mutant protein persists, to accumulate to high levels within the cell (step 5).

For several gene mutations that are the bases of human diseases, the mutant membrane or secretory protein product is sufficiently active to prevent the disease state, but the product is captured by the quality control system in the ER and never reaches its site of function. One of the most well characterized examples involves the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel protein whose functional loss underlies cystic fibrosis (reviewed by Welsh, 1994; Welsh and Smith, 1995). The most common disease-related mutation in CFTR is a deletion of Phe508 (F508). Typically, F508 protein never reaches the plasma membrane (Cheng et al., 1990; Lukacs et al., 1994) because it is captured by chaperones in the ER quality control system and is eventually degraded by proteasomes in the cytoplasmic compartment (Ward et al., 1995). Interestingly, a portion of F508 protein can achieve an active conformation and correct localization in the plasma membrane when cells are grown at reduced temperature (Denning et al., 1992) or when F508 protein is overexpressed (Cheng et al., 1995). Expression at lower temperatures would favor native folding (consider, for example, the correct folding of temperature-sensitive mutants at permissive temperatures), whereas overexpression would be expected to increase the amount of F508 protein that escapes the limiting quality control system. Therefore, a critical problem with F508 (and probably with several other disease-related alleles) is the inability of the protein to move beyond the ER quality control system.

1. Alzheimer's disease. A common marker of Alzheimer's disease, and perhaps the major cause of neurodegeneration associated with this condition, is the formation in brain tissues of amyloid plaques, which are principally composed of the amyloid- (A) protein (reviewed by Checler, 1995; Dickson, 1997; Selkoe, 1994, 1996, 1997). The amyloid precursor protein (APP), whose normal cellular function is unresolved, is a membrane protein and, distinctively, an intramembranous substrate for proteases that generate a characteristic set of fragments. A is one of these and consists of the amino-terminal 40 to 43 amino acids of APP.

2. Huntington's disease. Huntington's disease is one of a class of neurodegenerative conditions caused by aggregation of a poly-glutamine-containing protein (reviewed by Bates et al., 1997; Lunkes and Mandel, 1997). Although different gene products are involved in different disorders, a common genetic feature is that each locus contains CAG tandem repeats that have been expanded by a poorly understood mechanism. Because CAG is a glutamine codon, an in-frame CAG expansion results in a protein product with a corresponding extension of its polyglutamine (polyGln) tract. Beyond a critical length of approximately 40 glutamine residues, protein aggregation occurs that leads to pathological changes.

3. Prion diseases. Sheep scrapie, bovine spongiform encephalopathy, and human Cruetzfeldt-Jakob disease are transmissible neurodegenerative diseases; the infectious agent for each is thought to be a prion vector termed prion protein (PrP) (Prusiner and Scott, 1997). Although findings are still controversial (Caughey and Chesebro, 1997), prion vectors are thought to lack nucleic acids and to consist solely of an alternately folded form of a protein that is normally expressed in the host. Strong genetic evidence for a heritable prion-like protein has come from studies of certain non-Mendelian inheritance patterns in yeast (Lindquist, 1997; Wickner, 1994, 1996).

	V. Natural Products That Bind Chaperone Components

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Several natural products synthesized by fungi and bacteria bind with high affinity and specificity to chaperone components. It is likely that these compounds are evolutionarily adapted to deter predators or competitors, with chaperone proteins being effective toxicological targets.

The important immunosuppressant drugs cyclosporin A, FK506 (tacrolimus), and rapamycin (sirolimus) function by virtue of their binding to proteins in the two immunophilin families, i.e., the Cyps that bind cyclosporin A and the FKBPs that bind FK506 and rapamycin (for reviews, see Kay, 1996; Marks, 1996). In both immunophilin classes, the drug binds the PPIase active site, inhibiting enzymatic activity, but immunosuppression is not a result of PPIase inhibition. Instead, the drug-immunophilin complex forms a hybrid surface composed partly of protein and partly of a portion of the drug that extends from the binding pocket. This hybrid structure binds avidly to proteins regulating key steps in the activation of immune responses. Both cyclosporin-CypA and FK506-FKBP12 complexes bind to and inhibit calcineurin (Friedman and Weissman, 1991; Liu et al., 1991), a protein phosphatase whose activity is required for T cell activation. On the other hand, the rapamycin-FKBP12 complex, because of structural differences in the exposed regions of rapamycin and FK506 in FKBP12 complexes, fails to bind and inhibit calcineurin; instead, the rapamycin-FKBP12 complex binds a class of proteins termed target of rapamycin (TOR). Mammalian TOR, which was first identified in yeast cells (Heitman et al., 1991) and which has also been termed FKBP-rapamycin-associated protein (Brown et al., 1994) or rapamycin and FKBP12 target 1 (Sabatini et al., 1994), is involved in signaling the G₁ to S phase transition in cells; rapamycin treatment induces G₁ arrest of T cells. TOR proteins share homology with phosphoinositol 3-kinases, but mammalian TOR was recently shown to have protein kinase activity that recognizes a (Ser/Thr)-Pro motif similar to that recognized by mitogen-activated protein kinase (Brunn et al., 1997; Burnett et al., 1998). Therefore, binding of the rapamycin-FKBP12 complex to TOR may alter the ability of TOR to phosphorylate substrates and propagate growth signals.

Many compounds related to FK506 and rapamycin have been generated in hopes of reducing immunosuppressive side effects or discovering novel pharmacological uses. Many nonimmunosuppressive analogs (NIAs) have been identified that bind immunophilins and inhibit PPIase activity without promoting binding to calcineurin or TOR. As demonstrated most clearly by Snyder and colleagues (Steiner et al., 1997a,b), immunosuppressant agents and some NIAs have neurotrophic actions that may be therapeutically useful (reviewed by Sabatini et al., 1997). The major immunophilin targets for the NIAs are probably FKBP12 and CypA, both of which are expressed at high levels in neural tissues (Steiner et al., 1992) and are up-regulated in regenerating nerves (Lyons et al., 1995).

Although immunosuppressive and neurotrophic drug effects are predominantly mediated by FKBP12 and CypA, other immunophilin family members also bind FK506, cyclosporin A, and their respective analogs. The large-molecular size, TPR-containing family members that associate with Hsp90 and steroid receptor complexes bind drugs with lower affinities than the small family members, with no well defined biological consequences. In several reports (reviewed by Pratt and Toft, 1997), the effects of immunosuppressant drugs on steroid receptor assembly and function have been examined. The drugs have little effect on the structure and function of receptor complexes in cell-free studies, although drug-dependent effects (some seemingly contradictory) have been reported using intact cell assays. A problem with some intact cell studies of steroid signaling is that any drug effect is assumed to relate to receptor-associated immunophilins, when drug effects may in fact occur indirectly through other immunophilins (for example, alteration of protein phosphorylation patterns by inhibition of the phosphatase calcineurin) or through immunophilin-independent drug actions (such as drug competition at membrane efflux transporters, for which some steroids are substrates). Additional studies that should better define the potential activities of immunosuppressant drugs that are directly mediated by TPR-containing immunophilins are underway in several laboratories.

B. Molecules That Bind Hsp70

1. Background. Hsp70-class molecular chaperones perform numerous cellular functions, many of which appear to involve the binding and ATP-dependent release of nascent polypeptides or mature cellular proteins. As a result, it has been possible to identify synthetic peptides that bind selectively to Hsp70-class chaperones in the submillimolar concentration range; these reagents have been used in attempts to define the binding specificities of individual Hsp70 family members in vitro (Flynn et al., 1991; Fourie et al., 1994). Unfortunately, because of their inherent limitations in terms of rapid degradation in biological fluids, poor cellular uptake, and relatively low binding affinity, peptides have not proven useful in the study of Hsp70 function in intact cells. Several types of low-molecular weight compounds have been reported to modulate the expression and function of Hsp70-class molecules in whole cells, including drugs such as nonsteroidal anti-inflammatory agents (Morimoto et al., 1994), flavonoid kinase inhibitors (Elia et al., 1996), and serine/threonine phosphatase inhibitors such as okadaic acid (Chang et al., 1993). These drugs, however, are relatively nonspecific and appear to modulate the transcription of Hsp70, rather than interacting directly with the chaperone. In contrast, several derivatives of the natural product spergualin (an antibiotic that was first identified in culture filtrates of the organism Bacillus laterosporus) have been shown to interact selectively with an Hsp70 isoform and to alter its function (fig. 3). Spergualin is a peptidomimetic compound that contains the polyamine spermidine within its structure (Umezawa et al., 1981); it was first evaluated as an antitumor agent in murine leukemia models, where it exhibited significant activity (Takeuchi et al., 1981). Subsequently, the semisynthetic analog 15-deoxyspergualin (DSG) was noted to possess potent in vivo immunosuppressive activity, which was distinct from that of the classic immunophilin-binding drugs cyclosporin A and FK506.

View larger version (7K): [in this window] [in a new window]   Fig. 3.   Structure of DSG [7-[(aminoiminomethyl)amino]-N-[2-[[4-[(3-aminopropyl)amino]butyl]amino]-1-hydroxy-2-oxyethyl]heptanamide]. DSG is typically prepared as the trihydrochloride salt, with a formula weight of 497.

2. Hsp70-15-deoxyspergualin interactions. In an attempt to elucidate the mechanism of this novel immunosuppressive action, Nadler et al. (1992) prepared a solid-phase, immobilized, methoxy-DSG derivative to identify the cellular target(s) with which the drug interacted. Surprisingly, they found that a single major protein with an apparent molecular size of 70 kDa was able to bind selectively to DSG in lysates prepared from a variety of cell types. This protein could be eluted with soluble DSG or ATP but not with the polyamines putrescine and spermidine, which show some structural similarity to DSG but are not immunosuppressive. Peptide sequencing data, as well as immunoblotting with a panel of monoclonal antibodies, identified the DSG-interacting protein as Hsc70, the constitutive or cognate member of the Hsp70 class of Hsps. No binding of the inducible isoform of Hsp70 was detected but, because this species is present in much smaller amounts than Hsc70 under nonstress conditions, it is possible that it and other Hsps may also bind DSG. In fact, using affinity capillary electrophoresis and solution-phase DSG, Nadeau et al. (1994) found that DSG bound purified Hsc70 (K_d = 4 µM) and Hsp90 (K_d = 5 µM) with comparable affinities in Tris/glycine buffer. Given the typical concentrations of cytosolic Hsc70 (5 µM) and Hsp90 (2 to 10 µM) in cells, these K_d values for DSG interactions with Hsps are consistent with the concentrations required for biological activity in cell cultures and in vivo. This finding and the good correlation between immunosuppressive activity and the ability to bind Hsc70 for a panel of five DSG analogs suggested that Hsp function is actually a primary site of drug action. It is unclear why initial solid-phase affinity precipitation studies did not detect Hsp90 binding, but it is possible that Hsp90 binds to a site on DSG that was attached to the bead matrix or was close enough to result in sufficient steric hindrance to impede binding. DSG does not appear to interfere directly with Hsp90-Hsc70 interactions in vitro, and it does not inhibit the reconstitution of functional steroid receptors in reticulocyte lysates (Smith DF, unpublished observations). DSG binding to Hsc70 at low concentrations was found to stimulate the ATPase activity of Hsc70 2-fold, whereas at high concentrations activity returned to basal levels. Consistent with this finding, it was reported more recently that DSG interacts with Hsc70 in a fashion distinct from that of unfolded peptides (Nadler et al., 1995). Specifically, DSG was ineffective at inhibiting the binding of a cytochrome c peptide to purified Hsc70, and this peptide was unable to elute Hsc70 from DSG affinity resin. These observations are reminiscent of a report by Takenaka et al. (1995) describing the use of a phage display library to screen for Hsc70-interacting peptides. Those investigators found that Hsc70 recognized two distinct peptide motifs, one thought to be involved in Hsc70 chaperoning of proteins to organelles (NIVRKKK-like sequences) and one involved in Hsc70 facilitation of protein folding (FYQLALT-like sequences). The peptidomimetic structure of DSG may well interact with the hydrophobic-basic peptide binding site of Hsc70, which was shown to bind NIVRKKK sequences, but not the unfolded peptide binding site of Hsc70. Such an interaction could explain the lack of DSG effects on general protein folding while accounting for DSG interference with the chaperoning activity of Hsc70 that is potentially required for antigen processing and elaboration of cellular immune responses (see Section VI.C.).

3. 15-Deoxyspergualin biological activities. DSG was initially identified as a potential antitumor agent because of its activity against several mouse leukemia cell lines in vitro and in syngeneic animal models. It now appears that the cytotoxic activity of DSG in vitro is attributable in large part to its metabolism to aminoaldehydes and hydrogen peroxide by copper amine oxidases, which are present at high levels in fetal bovine serum (Shiro et al., 1992). When cell survival assays are performed in the presence of 1 mM aminoguanidine as an inhibitor of amine oxidase activity or in media containing sera that are low in oxidase activity (horse, mouse, or human serum), DSG appears to exert cytostatic effects that are characterized by G₁ cell cycle arrest (Nishikawa et al., 1991). Despite the demonstrated interaction of the drug with both Hsc70 and Hsp90, as discussed above, DSG does not revert the phenotype of cells transformed by activated tyrosine kinases such as v-Src, which are known to interact with these chaperones (Whitesell L, unpublished observations). Because the antitumor activity of DSG is markedly reduced in immunocompromised mouse models, it has been postulated that this anticancer activity may be the result of alterations in host immune function, but this possibility has yet to be proven. Unlike the immunosuppressants cyclosporin A and FK506, DSG does not interact with P-glycoprotein and does not reverse the multidrug resistance phenotype (Holmes and Twentyman, 1995).

C. Molecules That Bind Hsp90

1. Background. The molecular chaperone Hsp90 plays an essential role in stress tolerance, de novo protein folding, and posttranslational regulation of the stability and function of many important cellular proteins, including steroid hormones, protein kinases, and molecules regulating the cell cycle and programmed cell death. Although Hsp90 knockout is clearly lethal in eukaryotic cells, no well defined biochemical activity has been established for this class of molecular chaperones. Specifically, a role for ATP binding and/or hydrolysis in Hsp90-mediated chaperoning events has remained controversial. Recently, several small-molecule natural products that appear to interact in a selective fashion with Hsp90 have been identified by affinity precipitation techniques. The effects of these compounds on Hsp90 function in a variety of in vitro and cell culture systems are finally beginning to indicate the specific role(s) of Hsp90 in multiprotein chaperone complexes.

2. Hsp90-15-deoxyspergualin interactions. As described in Section V.B.2., Nadeau et al. (1994), using affinity capillary electrophoresis techniques, reported that the immunosuppressant DSG can bind purified Hsp90. Using solid-phase immobilized DSG, however, an Hsp90-DSG interaction was not detected in whole-cell lysates (Nadler et al., 1992). This discrepancy may be the result of technical problems associated with the DSG immobilization strategy used or may indicate a lack of physiological significance for the interaction detected by affinity capillary electrophoresis. Because of the lack of detectable effects on well defined Hsp90-dependent processes such as PR assembly and v-Src-mediated transformation, DSG has not proven helpful in defining Hsp90 function.

3. Hsp90-benzoquinone ansamycin interactions. In the course of screening microbial fermentation products for anticancer activity, it was noted that the benzoquinone ansamycins herbimycin A (Omura et al., 1979), geldanamycin (GA) (DeBoer et al., 1970), and macbecin (Muroi et al., 1979) were able to revert the phenotype of cells transformed by the oncogenically activated tyrosine kinase v-Src (Uehara et al., 1986) (fig. 4). Transformation by many other dominantly acting oncogenes, such as erbB-2, bcr-abl, fps, ros, and yes, have also been reported to be reversed by noncytotoxic concentrations of herbimycin A (Okabe et al., 1992; Uehara et al., 1988). Surprisingly, mechanistic evaluation of this novel biological activity has revealed that the ansamycins possess no intrinsic kinase inhibitory activity; they appear to alter the stability and function of kinases indirectly. Using a solid-phase-immobilized GA derivative, it was found that GA interacts in a highly selective fashion with Hsps of the 90-kDa class and that this interaction disrupts Hsp90 association with mutant kinases such as v-Src, leading to loss of their transforming activity (Whitesell et al., 1994). Similarly, GA causes disruption of Hsp90 interactions with a variety of kinases (Hartson et al., 1996; Nair et al., 1996; Schulte et al., 1995, 1996; Stepanova et al., 1996).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Structures of the benzoquinone ansamycins under most active evaluation as modulators of Hsp90 function. The molecular weight of herbimycin A is 560.

4. Hsp90-radicicol interactions. Radicicol is a macrocyclic antibiotic that was originally isolated from cultures of the fungus Monosporium bonorden (Delmotte and Delmotte-Plaquee, 1953). Although radicicol (fig. 5) is structurally distinct from the benzoquinone ansamycins, it too was originally described as a tyrosine kinase inhibitor with the ability to revert the morphological changes of v-Src-transformed fibroblasts (Kwon et al., 1992). In addition to inhibiting transformation by activated tyrosine kinases, radicicol has been reported to revert Ras transformation, presumably through disruption of downstream signaling by Raf kinase, because no alteration in the level of GTP-bound Ras was found in treated cells (Kwon et al., 1995). It was recently demonstrated that radicicol specifically interacts with Hsp90 (Schulte et al., 1998; Sharma et al., 1998) and can compete with GA for binding to the amino-terminal domain of this chaperone (Schulte et al., 1998). Given these biochemical findings, it is not surprising that the drug appears to possess many of the same biological activities as the benzoquinone ansamycins in cell culture. Although radicicol appears to have the same cellular target of action as GA-like compounds, its distinct structure (including the absence of a quinone ring) may result in quite different patterns of toxicity and bioactivity in whole animals because of differences in metabolism and disposition, which remain to be explored.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Structure of radicicol [5-chloro-6-(7,8-epoxy-10-hydroxy-2-oxo-3,5-undecadienyl)--resorcylic acid µ-lactone]. The molecular weight is 365.