High-Content Imaging Platform to Discover Chemical Modulators of Plasma Membrane Rafts.

Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

The C99 domain of the amyloid precursor protein resides in the disordered membrane phase.

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by ß-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-ß (Aß) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, ∼90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yet-unidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.

Delivery of recombinant SARS-CoV-2 envelope protein into human cells.

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

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.

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.

Small molecule NPT-440-1 inhibits ionic flux through Aß1-42 pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aß) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aß pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aß1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aß1-42 ß-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aß1-42 could prevent amyloid pore-mediated AD pathogenesis.

All-d-Enantiomer of ß-Amyloid Peptide Forms Ion Channels in Lipid Bilayers.

Alzheimer's disease (AD) is the most common type of senile dementia in aging populations. Amyloid ß (Aß)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aß-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aß binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aß peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aß effects even in the absence of receptor-peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aß isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aß isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aß isomer channels, and the d-isomer channel conductance is blocked by Zn(2+), a known blocker of l-Aß isomer channels. MD simulations further verify formation of ß-barrel-like Aß channels with d- and l-isomers, illustrating that both d- and l-Aß barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca(2+) leaking, unregulated channels in AD, and suggest that Aß toxicity is mediated through a receptor-independent, nonstereoselective mechanism.

Atomic force microscopy and MD simulations reveal pore-like structures of all-D-enantiomer of Alzheimer's ß-amyloid peptide: relevance to the ion channel mechanism of AD pathology.

Alzheimer's disease (AD) is a protein misfolding disease characterized by a buildup of ß-amyloid (Aß) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aß peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aß peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aß peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aß-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM), we imaged the structures of both D- and L-enantiomers of the full length Aß(1-42) when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aß isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Previously, we have shown that D-Aß(1-42) channels conduct ions similarly to their L- counterparts. Taken together, our results support the direct mechanism of Aß ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aß interaction with membrane receptors.

Probing structural features of Alzheimer's amyloid-ß pores in bilayers using site-specific amino acid substitutions.

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-ß (Aß) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aß channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aß channel is an assembly of loosely associated mobile ß-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aß(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining ß-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a ß-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aß-dependent ion fluxes.

Mutant SOD1 forms ion channel: implications for ALS pathophysiology.

Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wildtype SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a "toxic channel" mechanism presents a new therapeutic direction for ALS research.

Antimicrobial properties of amyloid peptides.

More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-ß structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape ß-strand-turn-ß-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link.

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich ß-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-ß (Aß(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aß(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aß(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing ß-rich AMPs and amyloids such as Aß, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and ß-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.

ß-Barrel topology of Alzheimer's ß-amyloid ion channels.

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small ß-amyloid (Aß) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered ß-sheets where each monomer folds into ß-strand-turn-ß-strand and the monomers are stacked atop each other. In the membrane, Aß peptides are proposed to be ß-type structures. Experimental structural data available from atomic force microscopy (AFM) imaging of Aß oligomers in membranes reveal heterogeneous channel morphologies. Previously, we modeled the channels in a non-tilted organization, parallel with the cross-membrane normal. Here, we modeled a ß-barrel-like organization. ß-Barrels are common in transmembrane toxin pores, typically consisting of a monomeric chain forming a pore, organized in a single-layered ß-sheet with antiparallel ß-strands and a right-handed twist. Our explicit solvent molecular dynamics simulations of a range of channel sizes and polymorphic turns and comparisons of these with AFM image dimensions support a ß-barrel channel organization. Different from the transmembrane ß-barrels where the monomers are folded into a circular ß-sheet with antiparallel ß-strands stabilized by the connecting loops, these Aß barrels consist of multimeric chains forming double ß-sheets with parallel ß-strands, where the strands of each monomer are connected by a turn. Although the Aß barrels adopt the right-handed ß-sheet twist, the barrels still break into heterogeneous, loosely attached subunits, in good agreement with AFM images and previous modeling. The subunits appear mobile, allowing unregulated, hence toxic, ion flux.

Structural convergence among diverse, toxic beta-sheet ion channels.

Recent studies show that an array of beta-sheet peptides, including N-terminally truncated Abeta peptides (Abeta(11-42/17-42)), K3 (a beta(2)-microglobulin fragment), and protegrin-1 (PG-1) peptides form ion channel-like structures and elicit single channel ion conductance when reconstituted in lipid bilayers and induce cell damage through cell calcium overload. Striking similarities are observed in the dimensions of these toxic channels irrespective of their amino acid sequences. However, the intriguing question of preferred channel sizes is still unresolved. Here, exploiting ssNMR-based, U-shaped, beta-strand-turn-beta-strand coordinates, we modeled truncated Abeta peptide (p3) channels with different sizes (12- to 36-mer). Molecular dynamics (MD) simulations show that optimal channel sizes of the ion channels presenting toxic ionic flux range between 16- and 24-mer. This observation is in good agreement with channel dimensions imaged by AFM for Abeta(9-42), K3 fragment, and PG-1 channels and highlights the bilayer-supported preferred toxic beta-channel sizes and organization, regardless of the peptide sequence.

Antimicrobial protegrin-1 forms ion channels: molecular dynamic simulation, atomic force microscopy, and electrical conductance studies.

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a beta-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the beta-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.

Treponema denticola PrcB is required for expression and activity of the PrcA-PrtP (dentilisin) complex.

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5' of prcA-prtP. The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6xHis-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6xHis in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.

Truncated beta-amyloid peptide channels provide an alternative mechanism for Alzheimer's Disease and Down syndrome.

Full-length amyloid beta peptides (Abeta(1-40/42)) form neuritic amyloid plaques in Alzheimer's disease (AD) patients and are implicated in AD pathology. However, recent transgenic animal models cast doubt on their direct role in AD pathology. Nonamyloidogenic truncated amyloid-beta fragments (Abeta(11-42) and Abeta(17-42)) are also found in amyloid plaques of AD and in the preamyloid lesions of Down syndrome, a model system for early-onset AD study. Very little is known about the structure and activity of these smaller peptides, although they could be the primary AD and Down syndrome pathological agents. Using complementary techniques of molecular dynamics simulations, atomic force microscopy, channel conductance measurements, calcium imaging, neuritic degeneration, and cell death assays, we show that nonamyloidogenic Abeta(9-42) and Abeta(17-42) peptides form ion channels with loosely attached subunits and elicit single-channel conductances. The subunits appear mobile, suggesting insertion of small oligomers, followed by dynamic channel assembly and dissociation. These channels allow calcium uptake in amyloid precursor protein-deficient cells. The channel mediated calcium uptake induces neurite degeneration in human cortical neurons. Channel conductance, calcium uptake, and neurite degeneration are selectively inhibited by zinc, a blocker of amyloid ion channel activity. Thus, truncated Abeta fragments could account for undefined roles played by full length Abetas and provide a unique mechanism of AD and Down syndrome pathologies. The toxicity of nonamyloidogenic peptides via an ion channel mechanism necessitates a reevaluation of the current therapeutic approaches targeting the nonamyloidogenic pathway as avenue for AD treatment.

Misfolded amyloid ion channels present mobile beta-sheet subunits in contrast to conventional ion channels.

In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Abeta channels consist of the solid-state NMR-based U-shaped beta-strand-turn-beta-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that beta-sheet channels break into loosely associated mobile beta-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for beta-sheet channels formed by cytolytic PG-1 beta-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by beta-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting alpha-helices that robustly prevent ion leakage, rather than hydrogen-bonded beta-strands. The simulations suggest why conventional gated channels evolved to consist of interacting alpha-helices rather than hydrogen-bonded beta-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.

K3 fragment of amyloidogenic beta(2)-microglobulin forms ion channels: implication for dialysis related amyloidosis.

Beta(2)-microglobulin (beta(2)m) amyloid deposits are linked to dialysis-related amyloidosis (DRA) in hemodialysis patients. The mechanism by which beta(2)m causes DRA is not understood. It is also unclear whether only the full-length beta(2)m induces pathophysiology or if proteolytic fragments are sufficient for inducing this effect. Ser20-Lys41 (K3) is a digestion fragment of full-length beta(2)m. Solid state NMR (ssNMR) combined with X-ray diffraction and atomic force microscopy (AFM) revealed the characteristic oligomeric amyloid conformation of the U-turn beta-strand-turn-beta-strand motif stacked in parallel and stabilized by intermolecular interactions also shown by Abeta(9-40)/Abeta(17-42) and the CA150 WW domain. Here we use the K3 U-turn atomic coordinates and molecular dynamic (MD) simulations to model K3 channels in the membrane. Consistent with previous AFM imaging of other amyloids that show channel-like structures in the membrane, in the simulations K3 also forms ion channels with 3-6 loosely attached mobile subunits. We carry out AFM, single channel electrical recording, and fluorescence imaging experiments. AFM images display 3D ion channel topography with shapes, morphologies, and dimensions consistent with the theoretical model. Electrical conductance measurements indicate multiple single channel conductances, suggesting that various K3 oligomer sizes can constitute the channel structure. Fluorescence measurements in kidney cells show channel-mediated cell calcium uptake. These results suggest that the beta(2)m-induced DRA can be mediated by ion channels formed by its K3 fragment. Because the beta-strand-turn-beta-strand motif appears to be a universal amyloid feature, its ability to form ion channels further suggests that the motif may play a generic role in toxicity.