Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology.

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70-BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein.

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-∆SBD and Hsp70-∆NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD-substrate interaction also within the hydrophobic bilayer interior acyl chain region.

Protein-oxidized phospholipid interactions in cellular signaling for cell death: from biophysics to clinical correlations.

Oxidative stress is associated with several major ailments. However, it is only recently that the developments in our molecular level understanding of the consequences of oxidative stress in modifying the chemical structures of biomolecules, lipids in particular, are beginning to open new emerging insights into the significance of oxidative stress in providing mechanistic insights into the etiologies of these diseases. In this brief review we will first discuss the role of lipid oxidation in controlling the membrane binding of cytochrome c, a key protein in the control of apoptosis. We then present an overview of the impact of oxidized phospholipids on the biophysical properties of lipid bilayers and continue to discuss, how these altered properties can account for the observed enhancement of formation of intermediate state oligomers by cytotoxic amyloid forming peptides associated with pathological conditions as well as host defense peptides of innate immunity. In the third part, we will discuss how the targeting of oxidized phospholipids by i) pathology associated peptides and ii) host defense peptides can readily explain the observed clinical correlations associating Alzheimer's and Parkinson's diseases with increased risk for type 2 diabetes and age-related macular degeneration, and the apparent protective effect of Alzheimer's and Parkinson's diseases from some cancers, as well as the inverse, apparent protection by cancer from Alzheimer's and Parkinson's diseases. This article is part of a Special Issue entitled: Oxidized phospholipids-Their properties and interactions with proteins.

Class specific peptide inhibitors for secretory phospholipases A2.

Phospholipases A2 (PLA2) catalyze the hydrolytic cleavage of free fatty acids from the sn-2 OH-moiety of glycerophospholipids. These enzymes have a number of functions, from digestion to signaling and toxicity of several venoms. They have also been implicated in inflammation and are connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia. Accordingly, there is a keen interest to develop selective inhibitors for therapeutic use. We recently proposed a novel mechanism for the control of PLA2 activity with highly active protofibrils of PLA2 existing transiently before conversion to inactive amyloid fibrils [19]. In keeping with the above mechanism several algorithms identified (85)KMYFNLI(91) and (17)AALSYGFYG(25) in bee venom (bv) and human lacrimal fluid (Lf) PLA2, respectively, as a regions potentially forming amyloid type aggregates. Interestingly, in keeping with the proposed role of these sequences in the control of the activity of these enzymes, preincubation of 2nM bvPLA2 with (85)KMYFNLI(91) caused complete inhibition of PLA2 activity while the scrambled control peptide YNFLIMK had no effect. Approximately 36% attenuation of the hydrolytic activity of LfPLA2 present in human lacrimal fluid was observed in the presence of 80nM (17)AALSYGFYG(25).

Activation of phospholipase A2 by Hsp70 in vitro.

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer ß-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg(2+).

1-Palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, an oxidized phospholipid, accelerates Finnish type familial gelsolin amyloidosis in vitro.

Finnish type familial amyloidosis (FAF) is a neurodegenerative disease, which involves the deposition of D187N or -Y mutant gelsolin fragments as amyloid in various tissues, accompanied by dermatologic, neurologic, and ophthalmologic disorders. Like the other amyloid diseases, FAF is associated with oxidative stress. The latter results in an extensive chemical modification of biomolecules, such as the formation of a myriad of phospholipids with oxidatively modified acyl chains containing various functional groups. Here we demonstrate that 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde moiety at the end of its truncated sn-2 acyl chain, accelerates amyloidogenesis of FtG(179-194) (i.e., the core amyloidogenic segment of residues 179-194 of FAF gelsolin) as revealed by thioflavin T (ThT) fluorescence and electron microscopy. These techniques and Trp fluorescence show that the accelerated conversion of FtG(179-194) into amyloid fibrils consists of distinct consecutive phases. PoxnoPC at a close to critical micelle concentration (~22.5 µM) causes a maximal increase in ThT fluorescence and the K(app) for fibril formation. The rates of fibril elongation and nucleation were proportional to PoxnoPC concentration, while the rates of nucleation were different below and above the critical micelle concentration. Our data also suggest an initial rapid formation of a 1:1 complex by PoxnoPC and FtG(179-194). The latter could involve a transient Schiff base and reside at the membrane hydrocarbon-water interface in the proximity of the phosphocholine headgroup. Subsequently, these profibrils insert into a more hydrophobic milieu and undergo a slow structural transition and assemble into amyloid fibers. Different phases can be expected when proteins aggregate on the phospholipid membrane surfaces, underlying the importance of a detailed kinetic analysis to fully understand the effects of oxidized phospholipids on amyloidogenesis. This study represents the first comprehensive analysis of the kinetics and mechanisms of amyloid formation in the presence of an oxidized phospholipid.

Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine in vitro.

Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

Binding of amphipathic alpha-helical antimicrobial peptides to lipid membranes: lessons from temporins B and L.

Temporins constitute a family of amphipathic alpha-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic alpha-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies 'conformational switches', domains with equal probabilities for adopting random coil, alpha-helical and beta-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid beta-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.

High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidic acid.

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of X(PA) = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) forms amyloid-like fibrils by interaction with acidic phospholipids and inhibits Aß aggregation.

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) is a mammalian protein that is a member of the Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis related proteins group 1 (CAP) superfamily of proteins. A role for the common CAP domain in the function of the diverse superfamily members has not been described so far. Here, we show by a combination of independent techniques including electron microscopy, Thioflavin T fluorescence, and circular dichroism that GAPR-1 has the capability to form amyloid-like fibrils in the presence of liposomes containing negatively charged lipids. Surprisingly, GAPR-1 was also shown to bind the amyloid-oligomer specific antibody A11 in the absence of lipids, indicating that GAPR-1 has an intrinsic tendency to form oligomers. This behavior is characteristic for proteins that interfere with Aß aggregation and indeed we found that GAPR-1 effectively inhibited aggregation of Aß(1-40) peptide. Immuno-dot blot analysis revealed that GAPR-1 binds to prefibrillar oligomeric Aß structures during the early stages of fibril formation. Another CAP domain-containing protein, CRISP2, was also capable of forming fibrils, indicating that oligomerization and fibril formation is a shared characteristic between CAP family members. We suggest that the CAP domain may regulate protein oligomerization in a large variety of proteins that define the CAP superfamily.