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.

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-β (Aβ) peptides are the most likely causative agent. Aβ 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 Aβ intermediate aggregated states.

Modulation of Alzheimer's amyloid β 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 β (Aβ) peptides result in the production of a variety of aggregates that are neurotoxic.

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.

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

Interaction of the Alzheimer's Aβ 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 Aβ42 for several days.

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.

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

We performed a fluorescent analysis of the binding of Aβ 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 Aβ Competitive binding between Aβ 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 Aβ. We found that populations of cells that exhibited high surface membrane binding affinity for Aβ also show higher membrane cholesterol levels compared to cells that did not bind Aβ.

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

Although it is clear that amyloid beta (Aβ) peptides play a pivotal role in the development of Alzheimer's disease, the precise molecular model of action remains unclear. Aβ 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.

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.

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.

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.

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.

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.

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).

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.

Histidines 13 and 14 in the Abeta sequence are targets for inhibition of Alzheimer's disease Abeta ion channel and cytotoxicity.

The fact that Alzheimer's beta amyloid (Abeta) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Abeta ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Abeta channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations.

Early and late cytotoxic effects of external application of the Alzheimer's Abeta result from the initial formation and function of Abeta ion channels.

Extracellular application of the Alzheimer's beta-amyloid (Abeta) peptide evokes a series of cellular responses that leads to the death of cells by apoptosis. Some responses to freshly prepared Abeta occur immediately, including changes in intracellular calcium concentration and changes in membrane permeability and phosphatidylserine asymmetry. We show here that the cytotoxic action of externally applied Abeta, such as caspase activation and apoptotic loss of cell viability, occurs and persists even several days after Abeta is removed from the medium.

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.

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.

Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.

Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates.

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.

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.

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).