Anatoli B. Meriin

The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

ABSTRACT 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 cytochromec-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.

Hsp70 Prevents Activation of Stress Kinases A NOVEL PATHWAY OF CELLULAR THERMOTOLERANCE

Harmful conditions including heat shock, oxidative stress, UV, and so forth cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Here we report that elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduced p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhanced such activation. In addition, we have found that accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases, which can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a “sensor” of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells.

Proteasome Inhibitors Activate Stress Kinases and Induce Hsp72 DIVERSE EFFECTS ON APOPTOSIS

Inhibition of the major cytosolic protease, proteasome, has been reported to induce programmed cell death in several cell lines, while with other lines, similar inhibition blocked apoptosis triggered by a variety of harmful treatments. To elucidate the mechanism of pro- and antiapoptotic action of proteasome inhibitors, their effects on U937 lymphoid and 293 kidney human tumor cells were tested. Treatment with peptidyl aldehyde MG132 and other proteasome inhibitors led to a steady increase in activity of c-Jun N-terminal kinase, JNK1, which is known to initiate the apoptotic program in response to certain stresses. Dose dependence of MG132-induced JNK activation was parallel with that of apoptosis. Furthermore, inhibition of the JNK signaling pathway strongly suppressed MG132-induced apoptosis. These data indicate that JNK is critical for the cell death caused by proteasome inhibitors. An antiapoptotic action of proteasome inhibitors could be revealed by a short incubation of cells with MG132 followed by its withdrawal. Under these conditions, the major heat shock protein Hsp72 accumulated in cells and caused suppression of JNK activation in response to certain stresses. Accordingly, pretreatment with MG132 reduced JNK-dependent apoptosis caused by heat shock or ethanol, but it was unable to block JNK-independent apoptosis induced by TNFα. Therefore, proteasome inhibitors activate JNK, which initiates an apoptotic program, and simultaneously they induce Hsp72, which suppresses JNK-dependent apoptosis. A balance between these two effects might define the fate of cells exposed to the inhibitors.

Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72.

ABSTRACT Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.

Role of Hsp70 in regulation of stress-kinase JNK: implications in apoptosis and aging

Cell protection from stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from stresses which do not cause direct protein damage, e.g. some genotoxic agents. These stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a stress‐activated protein kinase, JNK, an early component of stress‐induced apoptotic signalling pathway. This finding provides the basis for the anti‐apoptotic activity of Hsp72. These observations can explain increased stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by stresses and subsequently to a higher rate of apoptotic death.

Aggregation of Expanded Polyglutamine Domain in Yeast Leads to Defects in Endocytosis

ABSTRACT The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.

The Function of HSP72 in Suppression of c-Jun N-terminal Kinase Activation Can Be Dissociated from Its Role in Prevention of Protein Damage

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.