Connecting Hsp70, sphingolipid metabolism and lysosomal stability

Heat shock protein 70 (Hsp70) is an evolutionary highly conserved molecular chaperone. Upon cancer-associated translocation to the lysosomal compartment, it promotes cell survival by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced death. We have recently shown that Hsp70 stabilizes lysosomes by binding to the endo-lysosomal lipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingolipid catabolism. The Hsp70–BMP interaction enhances the activity of acid sphingomyelinase, an important enzyme that hydrolyzes sphingomyelin. Importantly, treatment with recombinant Hsp70 effectively reverts the dramatic increase in lysosomal volume and decrease in lysosomal stability in cells from patients with Niemann-Pick disease, a genetic disorder associated with reduced acid sphingomyelinase activity. These findings give new insight into the mechanisms controlling lysosomal stability and integrity, and open new exciting possibilities for the treatment of cancer as well as Niemann-Pick disease.     

Hsp72 expression enhances survival in adenosine triphosphate-depleted renal epithelial cells

Although prior heat stress (HS) inhibits apoptosis in adenosine phosphate (ATP)-depleted renal epithelial cells (REC), the specific stress protein(s) responsible for cytoprotection have not been identified. The present study evaluated the hypothesis that Hsp72, the major inducible member of the Hsp70 family, protects REC against ATP depletion injury. In the presence of isopropyl-beta-D-thiogalactoside (IPTG), a stable line of transfected opossum kidney cells was induced to overexpress human Hsp72 tagged with the flag epitope. Transfected cells from 2 clones that expressed Hsp72 at a level comparable with wild-type cells were subjected to transient heat stress (43 degrees C for 1 hour). To assess the cytoprotective effect of Hsp72, transfected cells were subjected to transient ATP depletion followed by recovery in the presence vs the absence of IPTG. ATP depletion resulted in nuclear chromatin condensation without cell membrane injury (ie, minimal leak of lactate dehydrogenase) and activation of caspase-3, confirming that apoptosis is the major cause of cell death. In both clones cell survival 1-3 days after ATP depletion was significantly improved in the presence of IPTG. Selective overexpression of Hsp72 reproduced nearly 60% of the protective effect on the survival afforded by prior heat stress. In transfected cells subjected to ATP depletion, Hsp72 overexpression significantly inhibited caspase activation. In native renal cells brief ATP depletion markedly induced the expression of native Hsp72, a finding identical to that observed after renal ischemia in vivo. These studies are the first to directly show that Hsp72 per se mediates acquired resistance to ischemic injury in REC.     

Heat Shock Protein 70.1 (Hsp70.1) Affects Neuronal Cell Fate by Regulating Lysosomal Acid Sphingomyelinase

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion.     

Mechanisms for Hsp70 secretion: crossing membranes without a leader

Heat shock protein 70 (Hsp70) is released from cells of many types and plays a significant signaling role, particularly in the inflammatory and immune responses. However, Hsp70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. However, Hsp70 can be released from cells by active mechanism that are independent of de novo Hsp70 synthesis or cell death. This pathway is similar to one utilized by the leaderless protein interleukin 1beta. Hsp70 release involves transit through an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of Hsp70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of Hsp70 into this secretory compartment appears to involve the ABC-family transporter proteins. While the cell signals involved in triggering Hsp70 release through this lysosomal pathway are largely unknown, recent data suggest a regulatory role for extracellular ATP. These mechanisms are also shared by interleukin 1beta secretion. Following release it has been shown that Hsp70 binds to adjacent cells, suggesting that the secreted protein participates in paracrine or autocrine interactions with adjacent cell surfaces. Thus an outline is beginning to of the mechanisms of Hsp70 secretion. Much further study will be required to fully elucidate mechanisms involved in targeting Hsp70 towards the non-canonical secretion pathways and its regulation.     

Why are hippocampal CA1 neurons vulnerable but motor cortex neurons resistant to transient ischemia?

It is well-known that heat-shock protein 70.1 (Hsp70.1), a major protein of the human Hsp70 family, plays cytoprotective roles by both its chaperone function and stabilization of lysosomal membranes. Recently, we found that calpain-mediated cleavage of carbonylated Hsp70.1 in the hippocampal cornu Ammonis1 (CA1) contributes to neuronal death after transient global ischemia. This study aims to elucidate the differential neuronal vulnerability between the motor cortex and CA1 sector against ischemia/reperfusion. Fluoro-Jade B staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labeling analysis of the monkey brain undergoing 20min whole brain ischemia followed by reperfusion, showed that the motor cortex is significantly resistant to the ischemic insult compared with CA1. Up-regulation of Hsp70.1 but absence of its cleavage by calpain facilitated its binding with NF-κB p65/IκBα complex to minimize NF-κB p65 activation, which contributed to a neuroprotection in the motor cortex. In contrast, because activated μ-calpain cleaved carbonylated Hsp70.1 in CA1, the resultant Hsp70.1 dysfunction not only destabilized lysosomal membrane but also induced a sustained activation of NF-κB p65, both of which resulted in delayed neuronal death. We propose that the cascades underlying lysosomal stabilization and regulating NF-κB activation by Hsp70.1 may influence neuronal survival/death after the ischemia/reperfusion.     

Anticancer drugs up-regulate HspBP1 and thereby antagonize the prosurvival function of Hsp70 in tumor cells

The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level than Hsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs than were those with a low ratio. Ectopic expression of HspBP1 enhanced this effect of anticancer drugs in a manner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs up-regulated HspBP1 expression, whereas prevention of such up-regulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs. Overexpression of HspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.     

Hsp70.1 and related lysosomal factors for necrotic neuronal death

Necrosis has long been considered accidental and uncontrolled, but during the last decade, it became clear that necrosis is also a well-orchestrated form of cell demise, being as well programmed as apoptosis. To explain the mechanism of neuronal necrosis after ischemia/reperfusion, the 'calpain-cathepsin hypothesis' formulated in 1998 postulates that the post-ischemic μ-calpain activation compromises integrity of the lysosomal membrane, thereby leading to cathepsin spillage. Another cause of the lysosomal rupture occurring during reperfusion is reactive oxygen species (ROS) that generate 4-hydroxy-2-nonenal (HNE) by oxidation of membrane fatty acids such as linoleic and arachidonic acids. HNE is an endogenous neurotoxin, because HNE-induced carbonylation of the substrate protein shows loss of its function. However, the molecular mechanisms of lysosomal membrane breakdown are still poorly understood; especially, the biochemical cascade how μ-calpain and ROS work together to disrupt lysosomal membrane has remained unclarified. Three independent proteomic analyses of cerebral ischemia, glaucoma, or mild cognitive impairment in primates have altogether suggested that the common substrate of calpain and/or ROS is heat-shock protein 70.1 (Hsp70.1; simply Hsp70, also called Hsp72 or HSPA1), a major protein of the human Hsp70 family. Hsp70.1 serves cytoprotective roles as a guardian of the lysosomal membrane integrity by assisting sphingomyelin degradation or maintaining proper protein folding and recycling as a chaperone. However, calpain-mediated cleavage of Hsp70.1, especially after its carbonylation because of the oxidative stresses, can induce lysosomal rupture. Furthermore, Hsp70.1 dysfunction activates nuclear factor-kappaB (NF-κB) signaling that can also promote neurodegeneration. By focusing on Hsp70.1 and related lysosomal factors, this review describes rationale of lysosomal destabilization and rupture for executing programmed neuronal necrosis.     

Hyperthermia assists survival of astrocytes from oxidative-mediated necrotic cell death.

In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.     

Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3

Background

Human tumors differ from normal tissues in their capacity to present Hsp70, the major stress-inducible member of the HSP70 family, on their plasma membrane. Membrane Hsp70 has been found to serve as a prognostic indicator of overall patient survival in leukemia, lower rectal and non small cell lung carcinomas. Why tumors, but not normal cells, present Hsp70 on their cell surface and the impact of membrane Hsp70 on cancer progression remains to be elucidated.

Methodology/Principal Findings

Although Hsp70 has been reported to be associated with cholesterol rich microdomains (CRMs), the partner in the plasma membrane with which Hsp70 interacts has yet to be identified. Herein, global lipid profiling demonstrates that Hsp70 membrane-positive tumors differ from their membrane-negative counterparts by containing significantly higher amounts of globotriaoslyceramide (Gb3), but not of other lipids such as lactosylceramide (LacCer), dodecasaccharideceramide (DoCer), galactosylceramide (GalCer), ceramide (Cer), or the ganglioside GM1. Apart from germinal center B cells, normal tissues are Gb3 membrane-negative. Co-localization of Hsp70 and Gb3 was selectively determined in Gb3 membrane-positive tumor cells, and these cells were also shown to bind soluble Hsp70-FITC protein from outside in a concentration-dependent manner. Given that the latter interaction can be blocked by a Gb3-specific antibody, and that the depletion of globotriaosides from tumors reduces the amount of membrane-bound Hsp70, we propose that Gb3 is a binding partner for Hsp70. The in vitro finding that Hsp70 predominantly binds to artificial liposomes containing Gb3 (PC/SM/Chol/Gb3, 17/45/33/5) confirms that Gb3 is an interaction partner for Hsp70.

Conclusions/Significance

These data indicate that the presence of Gb3 enables anchorage of Hsp70 in the plasma membrane of tumors and thus they might explain tumor-specific membrane localization of Hsp70.     

Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases.

The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.     

Calpain-mediated Hsp70.1 cleavage in hippocampal CA1 neuronal death

Necrotic neuronal death is recently known to be mediated by the calpain-cathepsin cascade from simpler organisms to primates. The main event of this cascade is calpain-mediated lysosomal rupture and the resultant release of lysosomal cathepsins into the cytoplasm. However, the in-vivo substrate of calpain for inducing lysosomal destabilization still remains completely unknown. The recent proteomics data using the post-ischemic hippocampal CA1 tissues and glaucoma-suffered retina from the primates suggested that heat shock protein (Hsp) 70.1 might be the in-vivo substrate of activated μ-calpain at the lysosomal membrane of neurons. Hsp70.1 is known to stabilize lysosomal membrane by recycling damaged proteins and protect cells from oxidative stresses. Here, we studied the molecular interaction between activated μ-calpain and the lysosomal Hsp70.1 in the monkey hippocampal CA1 neurons after the ischemia-reperfusion insult. Immunofluorescence histochemistry showed a colocalization of the activated μ-calpain and upregulated Hsp70.1 at the lysosomal membrane of the post-ischemic CA1 neurons. In-vitro cleavage assay of hippocampal Hsp70.1 by Western blotting demonstrated that Hsp70.1 in the CA1 tissue is an in-vivo substrate of activated μ-calpain, and that carbonylated Hsp70.1 in the CA1 tissue by artificial oxidative stressors such as hydroxynonenal (HNE) or hydrogen peroxide is much more vulnerable to the calpain cleavage. These data altogether suggested that Hsp70.1 can become a target of the carbonylation by HNE, and Hsp70.1 is a modulator of calpain-mediated lysosomal rupture/permeabilization after the ischemia-reperfusion injury.     

Proteomic identification of carbonylated proteins in the monkey hippocampus after ischemia–reperfusion

Reactive oxygen species (ROS) are known to participate in neurodegeneration after ischemia–reperfusion. With the aid of ROS, the calpain-induced lysosomal rupture provokes ischemic neuronal death in the cornu Ammonis (CA) 1 of the hippocampus; however, the target proteins of ROS still remain unknown. Here a proteomic analysis was done to identify and characterize ROS-induced carbonyl modification of proteins in the CA1 of the macaque monkey after transient whole-brain ischemia followed by reperfusion. We found that carbonyl modification of heat shock 70-kDa protein 1 (Hsp70-1), a major stress-inducible member of the Hsp70 family, was extensively increased before the neuronal death in the CA1 sector, and the carbonylation site was identified to be Arg469 of Hsp70-1. The CA1 neuronal death conceivably occurs by calpain-mediated cleavage of carbonylated Hsp70 that becomes prone to proteolysis with the resultant lysosomal rupture. In addition, the carbonyl levels of dihydropyrimidinase-like 2 isoform 2, glial fibrillary acidic protein, and β-actin were remarkably increased in the postischemic CA1. Therefore, ischemia–reperfusion-induced oxidative damage to these proteins in the CA1 may lead to loss of the neuroprotective function, which contributes to neuronal death.     

Beta-sheet-specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to HAMLET

As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET—a complex of partially unfolded alpha-lactalbumin and oleic acid—is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.     

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells

CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.     

Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance.     

Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation

Hsp70 overexpression can protect cells from stress-induced apoptosis. Our previous observation that Hsp70 inhibits cytochrome c release in heat-stressed cells led us to examine events occurring upstream of mitochondrial disruption. In this study we examined the effects of heat shock on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock caused a conformational change in Bax that leads to its translocation to mitochondria, stable membrane association, and oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. Hsp70 did not physically interact with Bax in control or heat-shocked cells, indicating that Hsp70 acts to suppress signals leading to Bax activation. Hsp70 inhibited stress-induced JNK activation and inhibition of JNK with SP600125 or by expression of a dominant negative mutant of JNK-blocked Bax translocation as effectively as Hsp70 overexpression. Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capability or cells treated with a small molecule activator of apoptosome formation, indicating that it is unable to prevent cell death after mitochondrial disruption and caspase activation have occurred. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from mitochondria. Hsp70, therefore, inhibits events leading up to mitochondrial membrane permeabilization in heat-stressed cells and thereby controls the decision to die but does not interfere with cell death after this event has occurred.     

Hispolon promotes MDM2 downregulation through chaperone-mediated autophagy

Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH₄Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.     

Brain-derived heat shock protein 70-peptide complexes induce NK cell-dependent tolerance to experimental autoimmune encephalomyelitis

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139-151 (PLP139-151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139-151 that correlated with elevated IFN-gamma and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139-151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.