Small molecule blockers of the Alzheimer Abeta calcium channel potently protect neurons from Abeta cytotoxicity.

Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Abeta). We have hypothesized that the intrinsic Abeta calcium channel activity of the oligomeric Abeta polymer may be responsible for the neurotoxic properties of Abeta, and that Abeta channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Abeta channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Abeta channel and protect neurons from Abeta toxicity. Both block the Abeta channel with similar potency (approximately 500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.

Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels.

Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.

Polyhistidine peptide inhibitor of the Abeta calcium channel potently blocks the Abeta-induced calcium response in cells. Theoretical modeling suggests a cooperative binding process.

On the basis of the consistent demonstrations that the Abeta peptide of Alzheimer's disease forms calcium permeant channels in artificial membranes, we have proposed that the intracellular calcium increase observed in cells exposed to Abeta is initiated by calcium fluxes through Abeta channels. We have found that a small four-histidine peptide, NAHis04, potently inhibits the Abeta-induced calcium channel currents in artificial lipid membranes. Here we report that NaHis04 also potently blocks the intracellular calcium increase which is observed in cells exposed to Abeta. PC12 cells loaded with Fura-2AM show a rapid increase in fluorescence and a rapid return to baseline after Abeta is added to the medium. This fluorescence change occurs even when the medium contains nitrendipine, a voltage-gated calcium channel blocker, but fails to occur when application of Abeta is preceded by addition of NAHis04. Steep dose-response curves of the percentage of responding cells and cell viability show that NAHis04 inhibits in the micromolar range in an apparently cooperative manner. We have developed numerous models of Abeta pores in which the first part of the Abeta sequence forms a large beta-barrel ending at His 13. We have modeled how up to four NAHis04 peptides may block these types of pores by binding to side chains of Abeta residues Glu 11, His 13, and His 14.

Models of membrane-bound Alzheimer's Abeta peptide assemblies.

Although it is clear that amyloid beta (Aß) peptides play a pivotal role in the development of Alzheimer's disease, the precise molecular model of action remains unclear. Aß peptide forms assemble both in aqueous solution and in lipid membranes. It has been proposed that deleterious effects occur when the peptides interact with membranes, possibly by forming Ca(2+) permeant ion channels. In the accompanying manuscript, we propose models in which the C-terminus third of six Aß42 peptides forms a six-stranded ß-barrel in highly toxic soluble oligomers. Here we extend this hypothesis to membrane-bound assemblies. In these Aß models, the hydrophobic ß-barrel of a hexamer may either reside on the surface of the bilayer, or span the bilayer. Transmembrane pores are proposed to form between several hexamers. Once the ß-barrels of six hexamers have spanned the bilayer, they may merge to form a more stable 36-stranded ß-barrel. We favor models in which parallel ß-barrels formed by N-terminus segments comprise the lining of the pores. These types of models explain why the channels are selective for cations and how metal ions, such as Zn(2+) , synthetic peptides that contain histidines, and some small organic cations may block channels or inhibit formation of channels. Our models were developed to be consistent with microscopy studies of Aß assemblies in membranes, one of which is presented here for the first time.

The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

Efficiency of histidine-associating compounds for blocking the alzheimer's Abeta channel activity and cytotoxicity.

The opening of the Alzheimer's Abeta channel permits the flux of calcium into the cell, thus critically disturbing intracellular ion homeostasis. Peptide segments that include the characteristic histidine (His) diad, His(13) and His(14), efficiently block the Abeta channel activity, blocking Abeta cytotoxicity. We hypothesize that the vicinal His-His peptides coordinate with the rings of His in the mouth of the pore, thus blocking the flow of calcium ions through the channel, with consequent blocking of Abeta cytotoxicity. To test this hypothesis, we studied Abeta ion channel activity and cytotoxicity after the addition of compounds that are known to have His association capacity, such as Ni(2+), imidazole, His, and a series of His-related compounds. All compounds were effective at blocking both Abeta channel and preventing Abeta cytotoxicity. The efficiency of protection of His-related compounds was correlated with the number of imidazole side chains in the blocker compounds. These data reinforce the premise that His residues within the Abeta channel sequence are in the pathway of ion flow. Additionally, the data confirm the contribution of the Abeta channel to the cytotoxicity of exogenous Abeta.

The cell-selective neurotoxicity of the Alzheimer's Abeta peptide is determined by surface phosphatidylserine and cytosolic ATP levels. Membrane binding is required for Abeta toxicity.

Measurement of Abeta toxicity of cells in culture exposes a subpopulation of cells with resistance to Abeta, even at high concentrations and after long periods of treatment. The cell-selective toxicity of Abeta resembles the selective damage observed in cells of specific regions of the Alzheimer's disease (AD) brain and suggests that there must be particular characteristics or stages of these cells that make them exceptionally sensitive or resistant to the effect of Abeta. Using flow cytometry and cell sorting, we efficiently separated and analyzed the Abeta-sensitive and the Abeta-resistant subpopulations within a variety of neuronal cell lines (PC12, GT1-7) and primary cultured neurons (hippocampal, cortex). We found that this distinctive sensitivity to Abeta was essentially associated with cell membrane Abeta binding. This selective Abeta binding was correlated to distinctive cell characteristics, such as cell membrane exposure of the apoptotic signal molecule phosphatidyl serine, larger cell size, the G1 cell cycle stage, and a lower than normal cytosolic ATP level. The response to Abeta by the cells with high Abeta binding affinity was characterized by a larger calcium response and increased mortality, lactate dehydrogenase release, caspase activation, and DNA fragmentation. The distinctive sensitivity or resistance to Abeta of the different subpopulations was maintained even after multiple cell divisions. We believe that these distinctive cell characteristics are the determining factors for the selective attack of Abeta on cells in culture and in the AD brain.

Abeta ion channels. Prospects for treating Alzheimer's disease with Abeta channel blockers.

The main pathological features in the Alzheimer's brain are progressive depositions of amyloid protein plaques among nerve cells, and neurofibrillary tangles within the nerve cells. The major components of plaques are Abeta peptides. Numerous reports have provided evidence that Abeta peptides are cytotoxic and may play a role in the pathogenesis of AD. An increasing number of research reports support the concept that the Abeta-membrane interaction event may be followed by the insertion of Abeta into the membrane in a structural configuration which forms an ion channel. This review summarizes experimental procedures which have been designed to test the hypothesis that the interaction of Abeta with a variety of membranes, both artificial and natural, results in the subsequent formation of Abeta ion channels We describe experiments, by ourselves and others, that support the view that Abeta is cytotoxic largely due to the action of Abeta channels in the cell membrane. The interaction of Abeta with the surface of the cell membrane may results in the activation of a chain of processes that, when large enough, become cytotoxic and induce cell death by apoptosis. Remarkably, the blockage of Abeta ion channels at the surface of the cell absolutely prevents the activation of these processes at different intracellular levels, thereby preserving the life of the cells. As a prospect for therapy for Alzheimer's disease, our findings at cellular level may be testable on AD animal models to elucidate the potential role and the magnitude of the contribution of the Abeta channels for induction of the disease.

Memory Loss and the Onset of Alzheimer's Disease Could Be Under the Control of Extracellular Heat Shock Proteins.

Alzheimer's disease (AD) is a major contemporary and escalating malady in which amyloid-ß (Aß) peptides are the most likely causative agent. Aß peptides spontaneously tend to aggregate in extracellular fluids following a progression from a monomeric state, through intermediate forms, ending in amyloid fibers and plaques. It is generally accepted now that the neurotoxic agents leading to cellular death, memory loss, and other AD characteristics are the Aß intermediate aggregated states. However, Aß peptides are continuously produced, released into the extracellular space, and rapidly cleared from healthy brains. Coincidentally, members of the heat shock proteins (hsp) family are present in the extracellular medium of healthy cells and body fluids, opening the possibility that hsps and Aß could meet and interact in the extracellular milieu of the brain. In this perspective and reflection article, we place our investigation showing that the presence of Hsp70s mitigate the formation of low molecular weight Aß peptide oligomers resulting in a reduction of cellular toxicity, in context of the current understanding of the disease. We propose that it may be an inverse relationship between the presence of Hsp70, the stage of Aß oligomers, neurotoxicity, and the incidence of AD, particularly since the expression and circulating levels of hsp decrease with aging. Combining these observations, we propose that changes in the dynamics of Hsp70s and Aß concentrations in the circulating brain fluids during aging defines the control of the formation of Aß toxic aggregates, thus determining the conditions for neuron degeneration and the incidence of AD.

Modulation of Alzheimer's amyloid ß peptide oligomerization and toxicity by extracellular Hsp70.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia caused by advanced neuronal dysfunction and death. The most significant symptoms of AD are observed at late stages of the disease when interventions are most likely too late to ameliorate the condition. Currently, the predominant theory for AD is the "amyloid hypothesis," which states that abnormally increased levels of amyloid ß (Aß) peptides result in the production of a variety of aggregates that are neurotoxic. The specific mechanisms for Aß peptide-induced cytotoxicity have not yet been completely elucidated. However, since the majority of Aß is released into the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells and makes its way inside where it disrupts the basic cellular process resulting in cell death. There is increasing evidence that hsp, particularly Hsp70, are exported into the extracellular milieu by an active export mechanism independent of cell death. Therefore, both Aß peptides and Hsp70 may coexist in a common environment during pathological conditions. We observed that Hsp70 affected the Aß assembling process in vitro preventing oligomer formation. Moreover, the presence of Hsp70 reduced the Aß peptide-induced toxicity of cultured neurons (N2A cells). These results suggest a potential mechanism for the reduction of the detrimental effects of Aß peptides in AD.

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes.

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.

Plasma membrane cholesterol controls the cytotoxicity of Alzheimer's disease AbetaP (1-40) and (1-42) peptides.

Cell degeneration in Alzheimer's disease is mediated by a toxic mechanism that involves interaction of the AbetaP peptide with the plasma membrane of the target cell. We report here that PC12 cells become resistant to the cytotoxic action of AbetaP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of AbetaP. Increasing cholesterol content of PS liposomes also suppresses AbetaP-dependent liposome aggregation. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol modulates the incorporation and pore formation of AbetaP into cell membranes. This idea is supported by our finding that the enhanced cytotoxicity generated by lowering the membrane cholesterol content can be reversed by AbetaP calcium channel blockers Zn2+ and tromethamine.

Hsc70 and Hsp70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability.

Heat shock proteins (hsps) are involved in multiple cellular processes during normal and stress conditions, particularly in the folding of polypeptides. A newly recognized property of the members of the Hsp70 family is their ability to interact with lipids, opening ion conductance pathways in artificial membranes, and integrating into natural membranes. The formation of Hsp70 channels in biological membranes and their function is still elusive. In this study, we showed that Hsp70 and Hsc70 display a highly selective interaction with phosphatidylserine moieties on membranes, followed by rapid incorporation into the lipid bilayer. Addition of Hsp70 or Hsc70 into the extracellular medium resulted in a viability decrease of cells beading PS on the exterior surface, such as PC12 cells. This toxic effect is modulated by the presence of ATP or ADP and can be blocked by screening PS moieties with annexin 5. These observations suggest that the presence of Hsp70 in the extracellular medium may be an accelerator of apoptosis since the presence of PS on the surface is an early indicator of this process. These findings may also explain the toxicity observed in cells overexpressing Hsp70s and provide a rational for the tight regulation of Hsp70 expression.

Single-cell screening of cytosolic [Ca(2+)] reveals cell-selective action by the Alzheimer's Aß peptide ion channel.

Interaction of the Alzheimer's Aß peptides with the plasma membrane of cells in culture results in chronic increases in cytosolic [Ca(2+)]. Such increases can cause a variety of secondary effects leading to impaired cell growth or cell degeneration. In this investigation, we made a comprehensive study of the changes in cytosolic [Ca(2+)] in single PC12 cells and human neurons stressed by continuous exposure to a medium containing Aß42 for several days. The differential timing and magnitude of the Aß42-induced increase in [Ca(2+)] reveal subpopulations of cells with differential sensitivity to Aß42. These results suggest that the effect produced by Aß on the level of cytosolic [Ca(2+)] depends on the type of cell being monitored. Moreover, the results obtained of using potent inhibitors of Aß cation channels such as Zn(2+) and the small peptide NA7 add further proof to the suggestion that the long-term increases in cytosolic [Ca(2+)] in cells stressed by continuous exposure to Aß is the result of Aß ion channel activity.

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70.

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.

Aggregation of liposomes induced by the toxic peptides Alzheimer's Abetas, human amylin and prion (106-126): facilitation by membrane-bound GM1 ganglioside.

To compare both the peptide molecular self-aggregation and the interaction with membrane lipids of the Alzheimer's amyloid beta (Abeta)40, Abeta42 peptides, and the cytotoxic peptides human amylin and prion (106-126) peptides, we applied a liposome aggregation technology. The kinetics of the changes in the optical density (DeltaOD) of liposome suspensions generated by the aggregation of liposomes induced by these peptides, allowed us to comparatively analyze their phospholipid affinity and self-aggregation. The kinetic curves showed an initial nonlinear region where d(DeltaOD)/dt followed first order kinetics corresponding to the binding of the peptides to the membrane of the liposome, a linear region where d(DeltaOD)/dt was constant, corresponding to the interaction between two membrane-bound peptide molecules, and a final slower increasing nonlinear region that corresponds to nucleation or seeding of aggregation. The analysis of the aggregation curves demonstrated that amylin and prion peptides also showed affinity for the acidic phospholipid phosphatidylserine (PS), as it has previously been shown for the Alzheimer's Abeta40, Abeta42 peptides. Abeta42 showed the highest, and amylin the lowest, affinity for the liposome membrane. When bound to the membrane of the liposomes, all the peptides preserved the self-aggregation characteristics observed in solution. Aging the Abeta40 and Abeta42 peptide solutions that permit molecular self-aggregation reduced their capacity to induce liposome aggregation. The self-aggregation of membrane-bound prion molecules was several orders of magnitude higher than that observed for the other toxic peptides. Incorporation of the ganglioside GM1 into the membrane of liposomes enhanced the peptide-induced liposome aggregation. Kinetic analysis revealed that this enhancement was due to facilitation of the formation of bridges between membrane-bound peptide molecules, demonstrating that the peptide-membrane interaction and the peptide amyloidogenesis are independent functions performed at separate molecular regions.

Annexin 5 and apolipoprotein E2 protect against Alzheimer's amyloid-beta-peptide cytotoxicity by competitive inhibition at a common phosphatidylserine interaction site.

Amyloid-beta-protein (betaA/4, AbetaP) accumulates in the brains of patients with Alzheimer's disease (AD), regardless of genetic etiology, and is thought to be the toxic principle responsible for neuronal cell death. The varepsilon4 allele of apoE has been linked closely to earlier onset of AD and increased deposition of the amyloid peptide, regardless of the clinical status of AD, while the apoE varepsilon2 allele is generally protective. We have previously hypothesized that the cell target for amyloid peptide might be the apoptotic signal molecule phosphatidylserine (PS). We report here that annexin 5, a specific ligand for PS, not only blocks amyloid peptide AbetaP[1-40] cytotoxicity, but competitively inhibits AbetaP[1-40]-dependent aggregation of PS liposomes. In addition, we find that apoE2, but not apoE4, can not only perform the same protective effect on cells exposed to AbetaP[1-40], but can also competitively inhibit PS liposome aggregation and fusion by the amyloid peptide. Altogether, the in vivo and in vitro results reported here provide fundamental insight to the process by which amyloid targets specific neurons for destruction, and suggest that PS may be a surface "receptor" site for AbetaP binding. These results also provide a biochemical mechanism by which the apoE varepsilon2 allele, but not apoE varepsilon4, can be protective towards the incidence and progression of Alzheimer's disease.

Interaction of heat shock protein 70 with membranes depends on the lipid environment.

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.

Fluorescent analysis of the cell-selective Alzheimer's disease aß Peptide surface membrane binding: influence of membrane components.

We performed a fluorescent analysis of the binding of Aß to the surface membrane of different types of cells lines such as PC12, GT1-7, and ex vivo neurons. Analyses were performed on sorted cells with membrane bound Aß Competitive binding between Aß phosphatidyl serine- (PtdSer-) specific binder annexin V and an anti-PtdSer antibody provided compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aß. We found that populations of cells that exhibited high surface membrane binding affinity for Aß also show higher membrane cholesterol levels compared to cells that did not bind Aß. This direct relationship was upheld in cholesterol-enriched or cholesterol-depleted cell membranes. We conclude that the initial process for the cell-selective binding by Aß, to later conversion of elemental Aß units into larger structures such as fibrils or to the potentially toxic ion channel aggregates, is highly influenced by the membrane content of PtdSer and cholesterol in the cell surface membrane.

Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages.

Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.