The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/ JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.Cellular stress impairs numerous physiological functions, damages cellular structures, and can lead to cell death. Death often occurs through an ordered pathway of self-destruction termed apoptosis (reviewed in references 23, 25, 73, and 78). This physiological mechanism of cell removal is essential during embryogenesis and for the maintenance of appropriate cell numbers in all multicellular organisms but is also triggered when cells are confronted with such nonphysiological conditions as exposure to mild hyperthermia, anoxia, UV irradiation, or certain chemotherapeutic or noxious drugs. Deregulated apoptosis is the underlying basis of a number of human pathologies. For example, impaired apoptosis contributes to the accumulation of neoplastic cells in many cancers whereas several neurological and immunological disorders are characterized by excessive cell elimination. Consequently, there has been intense interest in the signaling mechanisms, effector molecules, and essential targets of this evolutionarily conserved process.The apoptotic process is characterized by a distinct sequence of morphological and biochemical events. Cont...

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.Protein-damaging stresses, such as exposure of cells to elevated temperatures, activate an adaptive response leading to the increased synthesis of a group of proteins that regulate protein-folding processes (reviewed in reference 43). Members of the hsp70 family of molecular chaperones recognize nonnative domains that are exposed during protein translation, membrane translocation, oligomerization, and ultimately degradation. The abundant cytoplasmic and nuclear protein hsc70 is assisted in this task by the highly inducible hsp70 protein, whose synthesis is controlled by the level of nonnative protein substrates. Conditions that alter protein structure can result in the exposure of hydrophobic regions that are normally buried within the molecule, leading to their aggregation and loss of function. The ability of hsp70 to compete for binding to these hydrophobic regions coupled with an ATP...

The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.

Protein-damaging stresses induce the expression of 'heat-shock proteins', which have essential roles in protecting cells from the potentially lethal effects of stress and proteotoxicity. These stress-protective heat-shock proteins are often overexpressed in cells of various cancers and have been suggested to be contributing factors in tumorigenesis. An underlying basis of oncogenesis is the acquisition and accumulation of mutations that provide the transformed cell with the combined characteristics of deregulated cell proliferation and suppressed cell death. Heat-shock proteins with dual roles as regulators of protein conformation and stress sensors may therefore have intriguing and central roles in both cell proliferation and apoptosis. It has been established that heat-shock proteins exhibit specificity to particular classes of polypeptide substrates and client proteins in vivo, and that chaperones can stabilize mutations that affect the folded conformation. Likewise, overexpression of chaperones has also been shown to protect cells against apoptotic cell death. The involvement of chaperones, therefore, in such diverse roles might suggest novel anticancer therapeutic approaches targeting heat-shock protein function for a broad spectrum of tumor types.