| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Vol. 50, Issue 4, 493-514, December 1998
Department of Pharmacology (D.F.S.), University of Nebraska Medical Center, Omaha, Nebraska; Department of Pediatrics (L.W., E.K.), Steele Memorial Children's Research Center, University of Arizona, Tucson, Arizona
I. Protein Folding in the Cell
II. Molecular and Chemical Chaperones
A. Heat Shock Response and Heat Shock Proteins
B. Chaperone-Mediated Nascent Chain Folding
C. Cytoplasmic Co-chaperones
D. Endoplasmic Reticulum: A Special Folding Environment
E. Cellular Chemicals That Favor Protein Folding
III. Chaperone-Mediated Regulation of Signal Transduction Pathways
IV. Protein Misfolding in Disease
A. Cancer and Inactive or Inappropriately Acting Mutant Proteins
B. Cystic Fibrosis and Diversion in Folding Pathways
C. Amyloid Diseases and Protein Aggregation
1. Alzheimer's disease.
2. Huntington's disease.
3. Prion diseases.
V. Natural Products That Bind Chaperone Components
A. Molecules That Bind Immunophilins
B. Molecules That Bind Hsp70
1. Background.
2. Hsp70-15-deoxyspergualin interactions.
3. 15-Deoxyspergualin biological activities.
C. Molecules That Bind Hsp90
1. Background.
2. Hsp90-15-deoxyspergualin interactions.
3. Hsp90-benzoquinone ansamycin interactions.
4. Hsp90-radicicol interactions.
5. Biological activities.
VI. Prospective Therapeutic Rationales That Involve Chaperones
A. Drugs Targeting Specific Chaperone Activities
1. Immunophilins.
2. Hsp70.
3. Hsp90.
B. Induction of Protein and Chemical Chaperones
1. Background.
2. Mechanisms for inducing chaperone activity.
3. Injury protection.
4. Enhanced utilization of mutant proteins.
C. Chaperones as Immunological Adjuvants
VII. Summary
Acknowledgments
References
 |
I. Protein Folding in the Cell |
|---|
 Top NextReferences
|
|---|
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 |
|---|
|
TopPreviousNextReferences
|
|---|
A. Heat Shock Response and Heat Shock Proteins
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.
|
B. Chaperone-Mediated Nascent Chain Folding
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.
C. Cytoplasmic Co-chaperones
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.
D. Endoplasmic Reticulum: A Special Folding Environment
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.
E. Cellular Chemicals That Favor Protein Folding
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 |
|---|
|
TopPreviousNextReferences
|
|---|
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.
|
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 |
|---|
|
TopPreviousNextReferences
|
|---|
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.
A. Cancer and Inactive or Inappropriately Acting Mutant Proteins
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.
|
B. Cystic Fibrosis and Diversion in Folding Pathways
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.
C. Amyloid Diseases and Protein Aggregation
Misfolded proteins can lead to intracellular aggregate formation, disruption of multiple cellular processes, and cell death. However, just as in heat shock and other proteotoxic conditions, the chaperone machinery is designed to prevent aggregation, reverse aggregation that occurs, and/or target misfolded proteins and aggregated complexes for proteolytic degradation. Overall, the chaperone machinery functions efficiently in this regard, but there are some situations in which protein aggregation is not prevented or remedied, resulting in cellular abnormalities. The following neurodegenerative diseases highlight the potential hazards of protein aggregation.
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.
42- and
43-amino acid peptides. Mutations in APP associated with Alzheimer's
disease all map within or adjacent to the A sequence. However, the
most common mutations associated with familial Alzheimer's disease are
in the genes for presenilin 1 or 2 (Hutton and Hardy, 1997), proteins
that somehow regulate proteolysis of APP (De Strooper et
al., 1998).
The carboxyl-terminal region of A exists in a -sheet
conformation, whereas a region toward the amino terminus can exist in
either an -helical or -sheet conformation (Soto et
al., 1995). Various factors, such as A mutations, can shift the
conformational equilibrium of the amino-terminal region toward a
-sheet conformation that favors aggregation of A peptides and
plaque formation. Once a seed aggregate has formed, equilibrium might
be shifted further toward -sheet conformations as monomers in the
transient -strand conformation are trapped by addition to
preexisting aggregates. A conformational shift toward
-sheets is a factor in other diseases involving protein aggregation.
In addition to promoting amyloid plaque formation, accumulation of A
may be involved in other neurotoxic mechanisms. In one recent study
(Yan et al., 1997), a cellular protein termed endoplasmic reticulum-associated amyloid -peptide binding protein, which is related to hydroxysteroid dehydrogenases, was shown to bind specifically to A; this protein's abundance and interactions with
A correlated positively with A-induced neurotoxicity. In another
report (Schulze et al., 1993), recombinant A was found to
interact specifically with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis. Conceivably, sequestration of
GAPDH and a deficit in GAPDH activity could result in cellular metabolic deficiencies and contribute to neuropathological changes, but
this remains to be demonstrated.
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.
) codes for huntingtin (Htg), a
350-kDa protein. Htg appears to be expressed in all tissues, not just
the brain, and embryonic lethality is observed in mice nullizygous for
the Htg gene (Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995). In normal individuals, the
amino-terminal region of Htg contains a tract of 6 to 39 glutamine
residues; in Huntington's disease, the tract is 35 to 180 residues
long, but 40 to 55 units are found in most cases. Recent evidence
(White et al., 1997) suggests that Htg is required for
neurogenesis but that pathological changes associated with expanded
polyGln tracts in Htg are the result of a gain of function rather than
loss of normal Htg function.
In one recent study (Scherzinger et al., 1997), Htg
fragments containing a range of polyGln lengths were generated.
Fragments with 20 or 30 glutamines failed to aggregate in an in vitro
assay, but fragments with 51 or more glutamines formed fibrillar
aggregates. As with A, the aggregating form of Htg is
conformationally distinct from nonaggregating protein and appears to be
enriched for -sheet structure. Expression of only the amino-terminal
portion of Htg, containing a 100- to 150-glutamine tract, generated
Huntington-like pathological markers and symptoms in transgenic mice
(Davies et al., 1997). Neuronal intranuclear inclusions were
formed that contained the Htg fragment as well as ubiquitin, suggesting
that there might be a defect in ubiquitin-mediated degradation of the fragment. Interestingly, Htg is a substrate for apopain, a protease involved in apoptotic pathways, and cleavage increases with the length
of the polyGln tract (Goldberg et al., 1996). Apopain
cleavage generates an amino-terminal, polyGln fragment similar to the
transgene product observed in the study by Davies et al.
(1997); therefore, generation of an amino-terminal Htg fragment may be
pathogenic in Huntington's disease. Similar to the A interaction
with GAPDH noted above, peptides and Htg fragments containing an
extended polyGln tract bound specifically to GAPDH (Burke et
al., 1996). Again, although it is tempting to speculate that loss
of GAPDH activity may be directly related to disease mechanisms, the
significance of GAPDH binding has not been resolved.
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).
). The normal function of the noninfectious
cellular form of PrP (PrPC) is poorly defined.
PrPC is a membrane protein found in many tissues,
not only in brain. PrPC has a half-life of 3 to 6 hours in cells and is sensitive to proteases in vitro. The infectious
form of PrP (termed PrPSc to reflect its role in
scrapie) undergoes little or no turnover in cells and is resistant to
proteolysis in vitro. PrPSc, but not
PrPC, forms amyloid fibrils associated with
scrapie, bovine spongiform encephalopathy, and Cruetzfeldt-Jakob
disease. Similar to A and the disease-associated form of Htg,
PrPSc has a much higher content of -sheet
structure than does the normal conformation in
PrPC (Pan et al., 1993), and a
recombinant amino-terminal fragment of PrP (142 amino acids) has been
shown to switch between -helical and -sheet conformations in a
pH-dependent manner (Mehlhorn et al., 1996; Zhang et
al., 1997a). PrPSc is thought to propagate
itself by inducing PrPC to change its
conformation to the protease-resistant form, which then participates in
amyloid formation. PrPSc appears to be required
for infectivity and formation of amyloid fibers, but it may not be
required for PrP-dependent neuropathological changes. Several years
ago, an observation was made that PrP synthesized in a cell-free system
can exist in alternate topological forms in ER membranes (Lopez
et al., 1990). Recently, it was shown (Hegde et
al., 1998) that transgenic mice expressing a PrP mutant that favors a greater cytoplasmic orientation of PrP domains developed a
neurodegenerative phenotype in the absence of protease-resistant PrPSc.
As recently discussed (Caughey and Chesebro, 1997), several questions
remain regarding the mechanism by which PrPC is
converted to PrPSc, as well as whether
PrPSc is the infectious agent or instead serves
as a highly protective reservoir for an unidentified virus. There are
some suggestions that conversion may be mediated by an additional
cellular protein, perhaps one of the molecular chaperones (Telling
et al., 1995). Interestingly, the chaperone Hsp104 has been
genetically (Chernoff et al., 1995) and biochemically
(Schirmer and Lindquist, 1997) linked to the function of prion-like
proteins in yeast. In addition, Hsp104 was recently found to interact
in vitro with the -sheet conformer of a PrP fragment (not the
-helical conformer) and with the Alzheimer's protein A (Schirmer
and Lindquist, 1997). Hsp104 and the bacterial chaperone GroEL were
found to enhance PrPSc-dependent conversion of
PrPC in vitro, whereas other protein chaperones
had no effect and chemical chaperones inhibited conversion (DebBurman
et al., 1997). Still, the biological role of chaperone
interactions in the pathogenesis of mammalian prion-based diseases
remains to be defined.
| |
V. Natural Products That Bind Chaperone Components |
|---|
|
TopPreviousNextReferences
|
|---|
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.
A. Molecules That Bind Immunophilins
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 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
(Kd = 4 µM) and Hsp90
(Kd = 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 Kd 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).
). The cellular mechanisms by which DSG achieves these
effects are not well understood, but it clearly acts in a manner
distinct from that of cyclosporin A and FK506. Unlike those agents, DSG
does not inhibit cytokine production. It is a rather poor inhibitor of
T cell proliferation but can partially inhibit mixed lymphocyte
reactions even when added after the initiation of the culture. DSG
strongly inhibits expression of the interleukin 2 receptor on activated
T cells (Ramos et al., 1996). DSG has also been reported to
exert significant effects on humoral immune function, including
inhibition of B cell differentiation and 
light chain expression at
the messenger ribonucleic acid level (Tepper et al., 1995).
This effect may be mediated via inhibition of the nuclear translocation
of transcription factor NF-B, which is required for light chain
expression. Interestingly, in that model system, DSG also inhibited the
nuclear translocation of Hsc70 that is normally observed after heat
shock (without producing gross changes in total nuclear protein
composition), leading the authors to speculate that DSG may interfere
with the postulated role of Hsc70 in chaperoning the energy-dependent
translocation of proteins through the nuclear pore (Yang and DeFranco,
1994). Lastly, experiments by Hoeger et al. (1994) suggest
that DSG may interfere with antigen processing by cells of the
monocyte/macrophage or antigen-presenting cell lineage. Pretreatment of
antigen-presenting cells but not T cells prevented proliferative
responses to conventional antigens such as tetanus toxoid (which
require processing and presentation), whereas responses to
superantigens were not affected. Such a mechanism for DSG action is
attractive in light of the Hsc70-binding activity of the drug, because
Hsp70 family members are known to play a role in the binding and
intracellular transport of antigenic peptides (Manara et
al., 1993; Tamura et al., 1997).
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) 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, 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.), 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 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.
; Miller
et al., 1994b), which was subsequently identified as Grp94,
an ER homolog of Hsp90. GA binding to Grp94 appears to disrupt
association of the receptor tyrosine kinase erbB-2 with this chaperone,
leading to destabilization of the kinase and loss of its transforming activity (Miller et al., 1994a). A study of
structure-activity relationships has demonstrated a strong correlation
between the biological effects of the benzoquinone ansamycins and their
ability to bind members of the Hsp90 family (An et al.,
1997).
More recently, two groups have confirmed GA interaction with Hsp90 by
establishing the crystal structures of Hsp90 fragments complexed with
either GA or ATP. Stebbins et al. (1997) used monomeric recombinant human Hsp90 fragments to define a GA binding pocket within
the amino terminus of the protein and speculated that this pocket
represents a physiological binding site for Hsp90 interactions with
partially folded polypeptide substrates. In that model, GA is proposed
to act as a competitive inhibitor of target-chaperone interactions. In
contrast, another group, using limited proteolysis of the yeast homolog
of Hsp90, were able to generate an amino-terminal fragment (residues 1 to 220) that they crystallized as a dimer in the presence of
Mg2+ and either adenosine diphosphate (ADP), ATP,
or adenosine-5'-O-(3-thio)triphosphate. That work
definitively identified an ADP/ATP binding site within the amino
terminus of Hsp90, which shares homology with the ATP binding site of
the bacterial type II topoisomerase DNA gyrase B protein (Prodromou
et al., 1997). The site also coincides structurally with the
GA binding site previously identified by Stebbins et al.
(1997), which indicates that it is not a peptide binding pocket, as
originally proposed. The crystallographic data did not definitively establish an intrinsic ATPase activity for Hsp90, but the conformations of the Hsp90 fragments used in this study did vary substantially in
their ATP- versus ADP-bound forms, in agreement with findings reported
in a previous biophysical study using native Hsp90 (Csermely et
al., 1993). Taken together, the structural findings reported above
and recent, well executed, biochemical studies have finally resolved
the longstanding controversy of ATP involvement in Hsp90 function
(Grenert et al., 1997; Sullivan et al., 1997),
and they convincingly demonstrate that GA acts as an Hsp90-specific ATP mimetic that alters the function of the chaperone.
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.
|
5. Biological activities.
Although the drugs were originally
described as selective inhibitors of tyrosine phosphorylation, the
biological activities of both radicicol and benzoquinone ansamycins
such as GA are best explained in terms of their high affinity
interaction with members of the Hsp90 class of molecular chaperones. In
the case of Raf-1 (Schulte et al., 1995, 1996) and both
receptor-linked (Chavany et al., 1996; Miller et
al., 1994a) and Src-family tyrosine kinases (Hartson et
al., 1996), it has become clear that the drugs do not directly
inhibit kinase activity but, rather, destabilize the kinase proteins
and stimulate their degradation. Destabilization occurs via stimulation
of the ubiquitin conjugation and proteasome-mediated degradation of the
target proteins (Mimnaugh et al., 1996; Sepp-Lorenzo et al., 1995). For many of these drug-sensitive targets,
stable association of the kinase with Hsp90 has been demonstrated by co-precipitation, but the way in which drug-induced alterations in
Hsp90 complex formation with the target result in its enhanced ubiquitin conjugation remains unclear at this time. GA interaction with
Hsp90 also explains some of the more recently reported biochemical activities of GA, such as inhibition of steroid hormone receptor function (Segnitz and Gehring, 1997; Whitesell and Cook, 1996) and
alteration of the conformation and stability of mutant p53 species
(Blagosklonny et al., 1995, 1996). The consequences of drug
interactions with Hsp90 are probably best characterized with respect to
the PR, because so much is known about the posttranslational chaperone
interactions that take place normally with this steroid receptor (Smith
et al., 1995). GA binding of Hsp90 both in vitro and in
intact cells does not inhibit Hsp90 association with the receptor
protein but, rather, inhibits the association of Hsp90 with the
co-chaperone p23 (Johnson and Toft, 1995). As a result, the formation
of a mature receptor heteroprotein complex that is competent to bind
hormone in a high affinity manner is blocked. Therefore, these
Hsp90-binding agents can function as nonclassical antagonists of
hormone action because they do not interact with the receptor protein
itself. Such a mechanism of action could prove useful in overcoming the
resistance to antiestrogen therapy that often results from acquired
mutations of the receptor protein in breast cancers treated with
conventional antagonists such as tamoxifen. On the other hand, global
disruption of steroid hormone signaling after systemic exposure to
these agents could lead to the toxicities characteristic of functional
adrenal insufficiency. Although preclinical studies have been reported
describing some preliminary toxicological data for GA and derivatives
in animals, no data have yet been reported that address these
specific issues.
, 1993), induction of Hsp
expression (Hegde et al., 1995; Murakami et al.,
1991), cytoprotection after hypoxic/ischemic injury (Conde et
al., 1997; Ohtsuki et al., 1996), induction of
differentiation (Honma et al., 1989; Kondo et
al., 1989; Shimada et al., 1995), and inhibition of
immune and inflammatory responses (Hwang et al., 1996; June et al., 1990; Nishiya et al., 1995). These
diverse effects are consistent with drug-mediated alterations in Hsp90
function rather than simply inhibition of tyrosine kinase activity, but
the precise mechanisms of drug action in each system have yet to be
defined. In particular, the possibility that some of these effects may be the result of drug-mediated alterations in the expression or function of other Hsps, either alone or in combination with Hsp90, cannot be ruled out.
A concern in the use of GA and similar Hsp90-binding drugs is the
potential for pleiotropic adverse effects resulting from disruption of
normal cellular folding processes, in both cytoplasm and the ER. GA
partially inhibited renaturation of firefly luciferase in a
reticulocyte lysate model for refolding of denatured proteins (Thulasiraman and Matts, 1996) and may have similar effects on multiple
Hsp90-dependent folding processes in the cytoplasmic compartment. As
noted above for erbB2, GA may also alter folding of secretory or
membrane proteins whose folding in the ER is dependent on Grp94.
| |
VI. Prospective Therapeutic Rationales That Involve Chaperones |
|---|
|
TopPreviousNextReferences
|
|---|
A. Drugs Targeting Specific Chaperone Activities
1. Immunophilins.
As discussed above, several natural products
that bind specifically to components of the chaperone machinery have
been identified. The immunosuppressants cyclosporin A and FK506 are two
notable examples that are currently being used in many transplantation procedures, as well as in the treatment of autoimmune and inflammatory diseases. These drugs or their analogs may also prove useful as neurotrophic agents. As noted, the primary protein effectors for immunosuppression and neurotrophic actions are CypA and FKBP12, but
other immunophilin family members are attractive pharmacological effectors. The involvement of Cyp40, FKBP52, and FKBP51 in multiple signal transduction pathways compels further research into their potential as therapeutic targets. There is currently a poor
understanding of the biological functions of the large immunophilins,
but it is reasonable to predict that drugs will be discovered or
designed that more effectively, and perhaps specifically, block actions of the large immunophilins in therapeutically important ways. 2. Hsp70.
DSG is the only drug currently under clinical
evaluation that is known to interact selectively with Hsp70-class
chaperones. Although the precise mechanism of the potent
immunosuppressive activity of DSG is still unclear, it appears to be
unique, because the toxicities of DSG do not overlap with those of
standard immunosuppressive agents. Early human trials have demonstrated
a favorable safety profile, and it appears that DSG may be able to
reverse renal allograft rejection episodes that are refractory to
conventional agents. Based on these considerations, the drug may prove
quite useful in combination-therapy regimens after solid-organ
transplants, but further study is required before its true utility can
be defined (Gores, 1996 3. Hsp90.
Drugs specifically binding Hsp90 hold promise as
inhibitors of signal transduction pathways influenced by Hsp90-target
protein interactions. As discussed above, GA and the benzoquinone
ansamycins have received considerable attention as potential anticancer
agents (Supko et al., 1995).). Preclinical studies have
demonstrated significant antitumor effects in several animal models,
with structure-activity data indicating much greater in vivo activity
for the 17-allylamino derivative of GA (17-AAG) (fig. 4) than for the
parent compound (Schnur et al., 1995). To overcome severe
solubility problems, 17-AAG has been formulated as a microdispersed
suspension in phospholipid. Pharmacokinetic data obtained in rodents
using this formulation indicate that plasma concentrations sufficient
for good bioactivity in vitro (>1 µM) can be achieved for >3 h
after bolus intravenous administration. Microdispersed 17-AAG is now
undergoing full Investigational New Drug-directed toxicological
evaluation, and it is anticipated that National Cancer
Institute-sponsored phase I clinical trials will be initiated with
patients with refractory malignancies after regulatory approval
(Sausville et al., 1997). Preliminary toxicological studies
have shown that 17-AAG is better tolerated than GA in rats and dogs,
with the dose-limiting toxicity being hepatic in dogs and renal in
rats. Hematopoietic toxicity was observed for 17-AAG but was not
dose-limiting (Page et al., 1997). The marked variations in
toxicity profiles among animal species and between GA and 17-AAG
suggest that drug metabolism, rather than intrinsic limitations
associated with drug-Hsp90 interactions, may be a significant component
of benzoquinone ansamycin toxicity in vivo. In this regard, several
proprietary derivatives of radicicol, a non-quinone-containing
structure that appears to interact with Hsp90 in a manner similar to
that of GA, are under development by the pharmaceutical company Kyowa
Hakko Kogyo. These compounds are said to demonstrate no hepatic or
renal toxicity at doses associated with good antitumor activity in
several human tumor xenograft models in mice (Akinaga S, personal communication).; Baler et
al., 1992, 1996; Nadeau et al., 1993; Nair et
al., 1996) and possibly contribute to repression of HSF1 activity.
In a simple model (Morimoto, 1993), the Hsps are competitively depleted
from HSF1-repressive interactions when misfolded substrates accumulate
after a proteotoxic insult. Activated HSF1 then increases transcription
from heat shock genes; as Hsp levels increase, HSF1 again becomes
repressed and Hsp production is curtailed. Unfortunately for
investigators hoping to understand stress responses, HSF1 regulation is
much more complex (Morimoto et al., 1996), but Hsp90 has
been clearly implicated as a participant. A common consequence of GA or
herbimycin A treatment of cells is the induction of Hsp70 that results
from drug-induced activation of HSF1. The exact mechanism by which the
Hsp90-binding drugs lead to HSF1 activation has not been determined, but it certainly correlates with a change in chaperone interactions with HSF1. Interestingly, another consequence of GA treatment is
activation of the unfolded protein response in ER that leads to
increased ER chaperone expression (Lawson et al., 1998).
This GA effect is perhaps mediated through Grp94-dependent regulation of this response or accumulation of misfolded proteins in the ER. As
discussed in the next section, the ability to elevate chaperone activity in cells may have several therapeutic uses.
B. Induction of Protein and Chemical Chaperones
1. Background.
It has been long recognized that heat-treated
or chemically stressed cells and tissues gain a tolerance to further
stressful insults. This conditioning results in large part from the
induction of Hsps and chemical chaperones that enhance the cellular
environment for protein folding and stability. A heat shock response is
observed in many tissues after an elevation of body temperature of a
few degrees, and fever may, in part, be an adaptation to naturally increase intracellular chaperone activity. In a rat model (Blake et al., 1991 2. Mechanisms for inducing chaperone activity.
There are
several means by which chaperones may be therapeutically induced. An
obvious one is mild heating of tissues, but localizing and controlling
temperature elevation within a narrow, therapeutically beneficial range
can be difficult. Recently, several pharmacological compounds that can
induce or enhance induction of Hsp synthesis were identified. As
previously noted, the benzoquinoid ansamycins herbimycin A and GA can
induce Hsp70 synthesis and activation of HSF1. These drugs and the
unrelated compound radicicol are now known to specifically bind Hsp90,
and it appears that their mechanism for activating HSF1 involves
disruption of a negative regulatory interaction between Hsp90 and HSF1.
These or similar drugs may prove to be effective for acute localized or
systemic induction of a heat shock response. Nonsteroidal
anti-inflammatory drugs have been found to potentiate the activation of
HSF1 and a heat shock response (Amici et al., 1995; Udelsman et al., 1993), behavioral
stress induces Hsp70 levels in adrenal and vascular tissues, a
biochemical response that is mediated by neurohormonal factors and
elevated blood pressure (Xu et al., 1996) and that is
attenuated with aging (Blake et al., 1991; Udelsman et
al., 1993). Recently, there has been considerable interest in the
possibility that prophylactic induction of chaperone activity may be
highly beneficial in several clinical situations.; Fawcett
et al., 1997; Jurivich et al., 1992, 1995; Lee
et al., 1995). Perhaps related to this drug action,
arachidonic acid can also potentiate HSF1 activation, decreasing the
temperature elevation needed for robust Hsp induction (Jurivich
et al., 1994). Because arachidonic acid is a key mediator
and precursor in inflammatory responses, its potentiation of HSF1
activation may be an adaptation to enhance chaperone activity within
cells near the site of injury or infection.
3. Injury protection.
Currie et al. (1988), using
rats whose body temperature was raised to 42°C, first demonstrated
that heat stress promotes recovery of heart tissues from ischemic
injury. This protection correlates with the time course of elevated
Hsp70 levels (Karmazyn et al., 1990; Yellon and Latchman,
1992), and transgenic mice overexpressing Hsp70 exhibit reduced
postischemic injury (Marber et al., 1995; Plumier et
al., 1995). Interestingly, Hsp70-transgenic mice are also
protected from ischemic injury in the brain (Plumier et al., 1997).
), has been
shown to protect cardiomyocytes in culture (Morris et al., 1996). Although it is difficult to predict when natural occlusion of
cardiac vessels may occur, induction of Hsps may be beneficial during
scheduled surgical procedures that can cause ischemic damage.
A common problem with balloon angioplasty is restenosis, i.e.,
reclosure of the artery resulting from smooth muscle cell proliferation in response to mechanical injury. In an in vitro model, pretreatment of
cells using heat treatment or incubation with chemical inducers of the
heat shock response reduced subsequent injury-induced smooth muscle
cell proliferation (Slepian et al., 1996). Further
investigation may show that chaperone-related pretreatment can reduce
postangioplasty restenosis. Similarly, recovery from several stressful
therapeutic procedures may be enhanced by induction of chaperone
activity that is appropriately timed and localized before the procedure.
4. Enhanced utilization of mutant proteins.
As discussed in
section IV with respect to the quality control machinery of the
ER, disrupted trafficking may be a greater problem than the
ultimate ability of a mutant protein to fold into a functional
conformation. There are several reports that chemical chaperones can
assist mutant proteins in passing ER quality control and continuing on
to be secreted or properly localized at membrane sites. The mutant
phenotype in cells expressing the F508 form of CFTR could be
corrected by treating cells with any of several chemical chaperones
(Brown et al., 1996; Sato et al., 1996),
including glycerol, trimethylamine N-oxide, and deuterated water. Welch
and colleagues have extended their study of chemical chaperones to
several additional systems involving misfolded proteins. Chemical
chaperones were shown to inhibit formation of
PrPSc (Tatzelt et al., 1996) and were
shown to complement, in cells grown at a nonpermissive temperature,
temperature-sensitive mutations in the tumor suppressor p53, oncogenic
v-Src, or a ubiquitin-activating enzyme (Brown et al.,
1997). In the latter study, chemical chaperones were able to promote
proper folding of nascent mutant polypeptides; once folded into a
native conformation, the mutant proteins were stable at nonpermissive
temperatures after removal of chemical chaperones. More than 30 years
ago, it was proposed that "osmotic remediation" could correct
mutant proteins (Hawthorne and Friss, 1964); possibly in less than
another 30 years, this principal will achieve clinical application.
C. Chaperones as Immunological Adjuvants
A basic intracellular function of Hsps is peptide chaperoning
within and across cellular compartments. An increasing body of data
indicate that Hsp-bound peptides are similar to those that bind major
histocompatibility complex (MHC) molecules. Hsp70 members may chaperone
potential MHC class I ligand peptides toward the transporter associated
with antigen processing. Grp94/Gp96 may help stabilize unfolded class I
chains and shuttle cytosolic peptides to newly synthesized class I
molecules in the ER.
The tumor-derived Hsps Hsp70 and Grp94 elicit tumor-specific
protective immunity in mice (Suto and Srivastava, 1995; Tamura et
al., 1997; Udono and Srivastava, 1993, 1994). These Hsps are not
antigenic themselves, but they act as carriers of immunogenic peptides.
Hsp70 and Grp94 preparations isolated from unrelated tumors or normal
murine tissues were ineffective in generating protection (Udono and
Srivastava, 1994). Murine depletion experiments have suggested that
Hsp-peptide complexes are taken up by host macrophages, which may
direct the associated peptides toward the endogenous (MHC class I)
presentation pathway (Suto and Srivastava, 1995; Udono and Srivastava,
1994). Grp94 from T cell lymphoma cells infected with the vesicular
stomatitis virus was associated with the immunodominant peptide of
vesicular stomatitis virus, which is naturally presented by H-2Kb class
I molecules (Nieland et al., 1996). Grp94-associated
peptides are not restricted to those that can bind to the particular
MHC class I alleles of the host cells but include a wider repertoire of
antigenic peptides (Arnold et al., 1995). It appears that
Grp94 can bind all peptides that enter the ER by either
transporter-associated antigen processing-dependent or -independent
mechanisms (Arnold et al., 1997).
There are potential advantages in using Hsp-peptide complexes generated
in vivo, rather than purified or synthetic peptides, as the source of
tumor antigens. Use of HSP-associated peptides may circumvent the need
to identify tumor-specific antigens. Furthermore, individual peptides
are restricted to specific MHC molecules, whereas peptides associated
with Hsp70 or Grp94 from tumors represent an aggregation of epitopes
corresponding to numerous human lymphocyte antigen specificities
(Arnold et al., 1995). Professional antigen-presenting cells
are capable of processing the appropriate peptides based on the MHC
restriction elements and presenting them to the responder human
lymphocyte antigen-matched T cells. Immunization of a tumor-bearing host with a specific tumor-associated antigen may induce an antitumor T
cell response. However, this may eventually lead to outgrowth of tumor
cells not expressing this particular antigen. Hsp-peptide complexes
generated in vivo may reduce immunological escape variants by providing
the entire antigenic repertoire of that tumor, resulting in the
generation of T effector cells directed against different tumor epitopes.
Numerous investigators have recently shown that complexes formed in
vitro between peptides and Hsps can also induce specific T cell
responses. Complexes of murine liver-derived Grp94 and Hsp70 with a
variety of immunogenic peptides induced potent, specific, cytotoxic T
cell responses against these peptides (Blachere et al.,
1997). Noncovalent complexes of mycobacterial Hsp70 and influenza A
nucleoprotein were effective in eliciting peptide-specific T helper
responses in mice (Roman and Moreno, 1996). Moreover, a large fragment
of ovalbumin covalently linked to mycobacterial Hsp70 acted as an
adjuvant in generating cytotoxic T cells and protected immunized mice
against a lethal challenge with transfected melanoma expressing
ovalbumin peptides (Suzue et al., 1997). The strategy of
using large protein fragments linked to Hsps may offer the advantage of
providing numerous peptides that may be processed and presented by the
highly diverse MHC molecules.
Transfer of genes encoding mycobacterial Hsps into mammalian tumors can
also increase the immunogenicity of the tumors. Transfection of
the mycobacterial Hsp65 gene into J774 murine macrophage tumor cells
abrogated their tumorigenicity in syngeneic and athymic murine hosts,
whereas preimmunization of syngeneic mice with J774-Hsp65 cells
conferred protection to challenges with wild-type tumor (Lukacs
et al., 1993). Similarly, in vivo mediated gene transfer of
mycobacterial Hsp65 into J774 tumors resulted in tumor regression in
both immunocompetent syngeneic mice and severe combined
immunodeficient mice (Lukacs et al., 1997). However,
complete tumor regression was evident only in immunocompetent mice,
confirming the role of T cells in tumor rejection.
Taken together, these reports strongly support the potential utility of Hsps as natural immunological adjuvants. Hsp-peptide complexes, Hsp-peptide fusion proteins, or Hsp gene-transfected cells may be effectively used to stimulate potent, durable, and specific T cell responses against tumor cells or virally infected cells.
| |
VII. Summary |
|---|
|
TopPreviousNextReferences
|
|---|
In this review, we have presented an overview of protein misfolding as a basis for disease and have provided a prospective look at pharmacological approaches that may ultimately help to prevent or resolve protein-folding problems. Much of this article has focused on the molecular and chemical chaperones that assist in protein-folding processes and may be helpful in alleviating conditions that result from misfolding. A textbook-length treatise would be needed to fully cover these subjects, and we apologize for our oversights and biases in selecting the topics, examples, and citations that appear here. Currently, there are few pharmacological therapies that directly address protein misfolding and chaperone activity, so much of our outlook is necessarily speculative. Although some of our prognostications may prove to be inaccurate or unfeasible, perhaps for reasons we should have recognized, we would be surprised if chaperone-targeting drugs, including ones unforeseen by us, are not added to the clinical arsenal in the near future.
| |
Acknowledgments |
|---|
|
TopPreviousNextReferences
|
|---|
The authors thank Len Neckers, William J. Welch, and the anonymous reviewers for their insightful suggestions on this manuscript. Work in the laboratories of the authors contributing to this manuscript was supported by National Institutes of Health Grants DK44923 (D.F.S.), DK48218 (D.F.S.), and CA69537 (L.W.) and American Cancer Society Grant IM785A (E.K.).
| |
Footnotes |
|---|
a Address for correspondence: David F. Smith, Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198-6260. E-mail: dfsmith{at}mail.unmc.edu.
| |
Abbreviations |
|---|
A, amyloid protein;
17-AAG, 17-allylaminogeldanamycin;
ADP, adenosine diphosphate;
APP, amyloid
precursor protein;
ATP, adenosine triphosphate;
CFTR, cystic fibrosis
transmembrane conductance regulator;
Cyp, cyclophilin;
DNA, deoxyribonucleic acid;
DSG, deoxyspergualin;
ER, endoplasmic reticulum;
FKBP, FK506-binding protein;
GA, geldanamycin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GR, glucocorticoid receptor;
HSF1, heat shock transcription factor 1;
Hsp, heat shock protein;
Htg, huntingtin;
MHC, major histocompatibility complex;
NIA, nonimmunosuppressive analog;
polyGln, polyglutamine;
PPIase, peptidylprolyl isomerase;
PR, progesterone receptor;
PrP, prion
protein;
PrPC, cellular form of prion protein;
PrPSc, infectious (scrapie) form of prion protein;
TCP, T-complex polypeptide;
TPR, tetratricopeptide repeat;
TOR, target of
rapamycin.
| |
References |
|---|
|
TopPrevious |
|---|
F508 cystic fibrosis transmembrane conductance regulator protein.
Cell Stress Chaperones
1:
117-125.[Medline]-amyloid precursor protein and its regulation in Alzheimer's disease.
J Neurochem
65:
1431-1444[Medline].F508-CFTR by overexpression.
Am J Physiol
268:
L615-L624
an overview, in
Guidebook to Molecular Cheperons and Protein Folding Factors (Gething M-J ed) pp 89-95, Oxford University Press, Oxford, UK.F508) occurs in the endoplasmic reticulum and requires ATP.
EMBO (Eur Mol Biol Organ) J
13:
6076-6086[Medline]. kinase with heat shock proteins in rabbit reticulocyte lysates.
J Biol Chem
267:
18160-18167B activation and tyrosine phosphorylation of JAK2 and the subsequent induction of nitric oxide synthase in C6 glioma cells.
FEBS Lett
371:
333-336[Medline].-helices into -sheets features in the formation of the scrapie prion proteins.
Proc Natl Acad Sci USA
90:
10962-10966-amyloid precursor protein.
J Neurochem
60:
1915-1922[Medline].-protein precursor and the mechanism of Alzheimer's disease.
Annu Rev Cell Biol
10:
373-403.-amyloid precursor protein and the genetics of Alzheimer's disease.
Cold Spring Harb Symp Quant Biol
61:
587-596-helical to -strand transition in the amino-terminal fragment of the amyloid -peptide modulates amyloid formation.
J Biol Chem
270:
3063-3067 light chain expression in 70Z/3 pre-B cells by blocking lipopolysaccharide induced NF-B activation.
J Immunol
155:
2427-2436[Abstract]. kinase and heat shock proteins in rabbit reticulocytes maturing during recovery from anemia.
Exp Cell Res
238:
273-282[Medline]. kinase to acquire and maintain an activatable conformation.
J Biol Chem
272:
11648-11656 peptide and mediates neurotoxicity in Alzheimer's disease.
Nature (Lond.)
389:
689-695[Medline].
0031-6997/98/504-0493$03.00/0
PHARMACOLOGICAL REVIEWS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
This article has been cited by other articles:
|
|
 |
|
  S. Ujino, S. Yamaguchi, K. Shimotohno, and H. Takaku Heat-shock Protein 90 Is Essential for Stabilization of the Hepatitis C Virus Nonstructural Protein NS3 J. Biol. Chem., March 13, 2009; 284(11): 6841 - 6846. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Singh and W. D. Kruger Functional Rescue of Mutant Human Cystathionine {beta}-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae J. Biol. Chem., February 13, 2009; 284(7): 4238 - 4245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Martins, J. L. Ordonez, A. Garcia-Sanchez, D. Herrero, V. Sevillano, D. Osuna, C. Mackintosh, G. Caballero, A. P. Otero, C. Poremba, et al. A Pivotal Role for Heat Shock Protein 90 in Ewing Sarcoma Resistance to Anti-Insulin-like Growth Factor 1 Receptor Treatment: In vitro and In vivo Study Cancer Res., August 1, 2008; 68(15): 6260 - 6270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Shames and J. D. Minna IP6K2 is a client for HSP90 and a target for cancer therapeutics development PNAS, February 5, 2008; 105(5): 1389 - 1390. [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Baumler, D. W. Nelson, D. M. Ney, and G. E. Groblewski Loss of exocrine pancreatic stimulation during parenteral feeding suppresses digestive enzyme expression and induces Hsp70 expression Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G857 - G866. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Baker Evolution of metamorphosis: role of environment on expression of mutant nuclear receptors and other signal-transduction proteins Integr. Comp. Biol., December 1, 2006; 46(6): 808 - 814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Hawtin Pharmacological Chaperone Activity of SR49059 to Functionally Recover Misfolded Mutations of the Vasopressin V1a Receptor J. Biol. Chem., May 26, 2006; 281(21): 14604 - 14614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hinzpeter, J. Lipecka, F. Brouillard, M. Baudoin-Legros, M. Dadlez, A. Edelman, and J. Fritsch Association between Hsp90 and the ClC-2 chloride channel upregulates channel function Am J Physiol Cell Physiol, January 1, 2006; 290(1): C45 - C56. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cortes and H. Kantarjian New Targeted Approaches in Chronic Myeloid Leukemia J. Clin. Oncol., September 10, 2005; 23(26): 6316 - 6324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Millson, A. W. Truman, V. King, C. Prodromou, L. H. Pearl, and P. W. Piper A Two-Hybrid Screen of the Yeast Proteome for Hsp90 Interactors Uncovers a Novel Hsp90 Chaperone Requirement in the Activity of a Stress-Activated Mitogen-Activated Protein Kinase, Slt2p (Mpk1p) Eukaryot. Cell, May 1, 2005; 4(5): 849 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Griffin, T. V. Valdez, and R. Mestril Radicicol activates heat shock protein expression and cardioprotection in neonatal rat cardiomyocytes Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1081 - H1088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bagatell and L. Whitesell Altered Hsp90 function in cancer: A unique therapeutic opportunity Mol. Cancer Ther., August 1, 2004; 3(8): 1021 - 1030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. G. Mimnaugh, W. Xu, M. Vos, X. Yuan, J. S. Isaacs, K. S. Bisht, D. Gius, and L. Neckers Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity Mol. Cancer Ther., May 1, 2004; 3(5): 551 - 566. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Thomas, J. Kiehn, H. A Katus, and C. A Karle Defective protein trafficking in hERG-associated hereditary long QT syndrome (LQT2): molecular mechanisms and restoration of intracellular protein processing Cardiovasc Res, November 1, 2003; 60(2): 235 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Beliakoff, R. Bagatell, G. Paine-Murrieta, C. W. Taylor, A. E. Lykkesfeldt, and L. Whitesell Hormone-Refractory Breast Cancer Remains Sensitive to the Antitumor Activity of Heat Shock Protein 90 Inhibitors Clin. Cancer Res., October 15, 2003; 9(13): 4961 - 4971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Nimmanapalli, E. O'Bryan, D. Kuhn, H. Yamaguchi, H.-G. Wang, and K. N. Bhalla Regulation of 17-AAG--induced apoptosis: role of Bcl-2, Bcl-xL, and Bax downstream of 17-AAG--mediated down-regulation of Akt, Raf-1, and Src kinases Blood, July 1, 2003; 102(1): 269 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ficker, A. T. Dennis, L. Wang, and A. M. Brown Role of the Cytosolic Chaperones Hsp70 and Hsp90 in Maturation of the Cardiac Potassium Channel hERG Circ. Res., June 27, 2003; 92 (12): e87 - e100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sathiyaa and M. M. Vijayan Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1508 - C1515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Rajamani, C. L. Anderson, B. D. Anson, and C. T. January Pharmacological Rescue of Human K+ Channel Long-QT2 Mutations: Human Ether-a-Go-Go-Related Gene Rescue Without Block Circulation, June 18, 2002; 105(24): 2830 - 2835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Aguilar-Mahecha, B. F. Hales, and B. Robaire Chronic Cyclophosphamide Treatment Alters the Expression of Stress Response Genes in Rat Male Germ Cells Biol Reprod, April 1, 2002; 66(4): 1024 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Lee, R. Sultana, G. Pertea, J. Cho, S. Karamycheva, J. Tsai, B. Parvizi, F. Cheung, V. Antonescu, J. White, et al. Cross-Referencing Eukaryotic Genomes: TIGR Orthologous Gene Alignments (TOGA) Genome Res., March 1, 2002; 12(3): 493 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Grammatikakis, A. Vultur, C. V. Ramana, A. Siganou, C. W. Schweinfest, D. K. Watson, and L. Raptis The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation J. Biol. Chem., March 1, 2002; 277(10): 8312 - 8320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Huang, N. F. Mivechi, and D. Moskophidis Insights into Regulation and Function of the Major Stress-Induced hsp70 Molecular Chaperone In Vivo: Analysis of Mice with Targeted Gene Disruption of the hsp70.1 or hsp70.3 Gene Mol. Cell. Biol., December 15, 2001; 21(24): 8575 - 8591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Jiang, C. A. Ballinger, Y. Wu, Q. Dai, D. M. Cyr, J. Hohfeld, and C. Patterson CHIP Is a U-box-dependent E3 Ubiquitin Ligase. IDENTIFICATION OF Hsc70 AS A TARGET FOR UBIQUITYLATION J. Biol. Chem., November 9, 2001; 276(46): 42938 - 42944. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Eickelberg, J. Geibel, F. Seebach, G. Giebisch, and M. Kashgarian K+-induced HSP-72 expression is mediated via rapid Ca2+ influx in renal epithelial cells Am J Physiol Renal Physiol, August 1, 2001; 281(2): F280 - F287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Tsukahara, T. Yoshioka, and T. Muraki Molecular and Functional Characterization of HSC54, a Novel Variant of Human Heat-Shock Cognate Protein 70 Mol. Pharmacol., April 13, 2001; 58(6): 1257 - 1263. [Abstract] [Full Text]
|
|
|
|
|
|
R. Nimmanapalli, E. O’Bryan, and K. Bhalla Geldanamycin and Its Analogue 17-Allylamino-17-demethoxygeldanamycin Lowers Bcr-Abl Levels and Induces Apoptosis and Differentiation of Bcr-Abl-positive Human Leukemic Blasts Cancer Res., March 1, 2001; 61(5): 1799 - 1804. [Abstract] [Full Text]
|
|
|
|
|
|
R. Bagatell, G. D. Paine-Murrieta, C. W. Taylor, E. J. Pulcini, S. Akinaga, I. J. Benjamin, and L. Whitesell Induction of a Heat Shock Factor 1-dependent Stress Response Alters the Cytotoxic Activity of Hsp90-binding Agents Clin. Cancer Res., August 1, 2000; 6(8): 3312 - 3318. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. G. An, T. W. Schulte, and L. M. Neckers The Heat Shock Protein 90 Antagonist Geldanamycin Alters Chaperone Association with p210bcr-abl and v-src Proteins before Their Degradation by the Proteasome Cell Growth Differ., July 1, 2000; 11(7): 355 - 360. [Abstract] [Full Text]
|
|
|
|
|
|
S. J. Felts, B. A. L. Owen, P. Nguyen, J. Trepel, D. B. Donner, and D. O. Toft The hsp90-related Protein TRAP1 Is a Mitochondrial Protein with Distinct Functional Properties J. Biol. Chem., February 4, 2000; 275(5): 3305 - 3312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhou, Q. Gong, and C. T. January Correction of Defective Protein Trafficking of a Mutant HERG Potassium Channel in Human Long QT Syndrome. PHARMACOLOGICAL AND TEMPERATURE EFFECTS J. Biol. Chem., October 29, 1999; 274(44): 31123 - 31126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Russell, S. R. Whitt, M.-S. Chen, and M. Chinkers Identification of Conserved Residues Required for the Binding of a Tetratricopeptide Repeat Domain to Heat Shock Protein 90 J. Biol. Chem., July 16, 1999; 274(29): 20060 - 20063. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
 |
 |
 |
|
 |
 |
 |
