Amyloid oligomers and their membrane toxicity - A perspective study

Review: 264 refs.

The molecular structures of amyloid fibers and oligomers are required in order to understand and control their formation. Yet, their partially disordered and polymorphic nature hinders structural analyses. Fortunately, short segments from amyloid proteins, which exhibit similar biophysical properties to the full-length proteins, also form fibrils and oligomers and their atomic structures can be determined. Here we describe experimental procedures used to assess fiber-forming capabilities of amyloid peptide segments and their crystallization.

Cryoprotective agent (CPA) toxicity is the most limiting factor impeding cryopreservation of critically needed tissues and organs for transplantation and medical research. This limitation is in part due to the challenge of rapidly screening compounds to identify candidate molecules that are highly membrane permeable and non-toxic at high concentrations. Such a combination would facilitate rapid CPA permeation throughout the sample, enabling ice-free cryopreservation with minimal toxicity. This study presents a new method for rapidly assessing the cell membrane permeability and toxicity of candidate CPAs. The new method enables ~ 100 times faster permeability measurement than previous methods, while also allowing assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 °C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation.

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol–AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol–AmB structures at the cell surface, preventing AmB cytotoxicity.

Some protein and peptide aggregates, such as those of amyloid-β protein (Aβ), are neurotoxic and have been implicated in several neurodegenerative diseases. Aβ accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer's disease. Although the presence of Aβ deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between Aβ secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that Aβ40 fibrils can be obtained by incubating seed-free Aβ40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection-absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel β-sheets, whereas self-assembled Aβ40 molecules formed antiparallel β-sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn Aβ40 Our findings indicate that Aβ generally self-assembles into antiparallel β-structures but can also form protofibrils with parallel β-sheets by interacting with ganglioside-bound Aβ. We concluded that by promoting the formation of parallel β-sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of Aβ fibrils. These results advance our understanding of ganglioside-induced Aβ fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer's disease.

Parkinson's disease is characterized by a progressive accumulation of α-synuclein (α-syn) aggregates in Lewy bodies, extracellular deposits found in the midbrain, hypothalamus, and thalamus. The rate of α-syn aggregation, as well as the secondary structure of α-syn oligomers and fibrils, can be uniquely altered by lipids. However, the role of saturation of fatty acids (FAs) in such lipids in the aggregation properties of α-syn remains unclear. In this study, we investigated the effect of saturation of FAs in phosphatidylcholine (PC) and cardiolipin (CL), as well as a mixture of these phospholipids on the rate of α-syn aggregation. We found that although saturation plays very little if any role in the rate of protein aggregation and morphology of α-syn aggregates, it determined the secondary structure of α-syn oligomers and fibrils. Furthermore, we found that aggregates formed in the presence of both saturated and unsaturated PC and CL, as well as mixtures of these phospholipids, exert significantly higher cell toxicity compared to the protein aggregates formed in the lipid-free environment. To extend these findings, we conducted in vivo studies using C. elegans, where we assessed the effect of lipid-modified α-syn aggregates on organismal survival and neurotoxicity. Our results suggest that the saturation of FAs in phospholipids present in the plasma and mitochondrial membranes can be a key determinant of the secondary structure and, consequently, the toxicity of α-syn oligomers and fibrils. These findings provide new insights into the role of lipids in Parkinson's disease pathogenesis and highlight potential targets for therapeutic intervention.

Thousands of antimicrobial peptides have been observed and studied in the past decades; however, their membrane-active mechanisms are ambiguous due to their dynamic structure in the cell membrane. Here, we applied both molecular dynamics (MD) simulations and biophysical experiments to study the small membrane-active antimicrobial peptide Hylaseptin P1 (HSP1), which has significant selectivity towards anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and bacterial model membranes. HSP1 does not bind and fold onto human red blood cell model membranes, and it only binds, but does not fold, in zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) membranes. This suggests that the lipid chemistry and membrane rigidity are key to prevent HSP1 binding onto membranes, and the lipid headgroup charge may further promote peptide folding in the membrane. Our experiment-validated MD simulations suggest a carpet-like model mechanism for HSP1 through peptide binding, folding, aggregation, and assembly. HSP1 is shorter than the membrane thickness; therefore, the folded peptides aggregate on the surface, cross the membrane, and the oligomeric structure is supported by several surface-bound peptides in both bilayer leaflets.

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Thermodynamics and kinetics analysis of lipid-assisted transport of intrinsically disorder proteins.

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein alpha-lactalbumin, known as human alpha-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4-30 lysozyme molecules. Each lysozyme molecule is able to bind 11-48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Collagen self-assembly and the development of tendon mechanical properties

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

IntroductionAlzheimer's disease (AD) is the most prevalent neurodegenerative disorder in elderly people. Existent therapies are directed at alleviating some symptoms, but are not effective in altering the course of the disease. MethodsBased on our previous study that showed that an Aβ-interacting small peptide protected against the toxic effects of amyloid-beta peptide (Aβ), we carried out an array of in silico, in vitro, and in vivo assays to identify a molecule having neuroprotective properties. ResultsIn silico studies showed that the molecule, referred to as M30 (2-Octahydroisoquinolin-2(1H)-ylethanamine), was able to interact with the Aβ peptide. Additionally, in vitro assays showed that M30 blocked Aβ aggregation, association to the plasma membrane, synaptotoxicity, intracellular calcium, and cellular toxicity, while in vivo experiments demonstrated that M30 induced a neuroprotective effect by decreasing the toxicity of Aβ in the dentate gyrus of the hippocampus and improving the alteration in spatial memory in behavior assays. DiscussionTherefore, we propose that this new small molecule could be a useful candidate for the additional development of a treatment against AD since it appears to block multiple steps in the amyloid cascade. Overall, since there are no drugs that effectively block the progression of AD, this approach represents an innovative strategy. SignificanceCurrently, there is no effective treatment for AD and the expectations to develop an effective therapy are low. Using in silico, in vitro, and in vivo experiments, we identified a new compound that is able to inhibit Aβ-induced neurotoxicity, specifically aggregation, association to neurons, synaptic toxicity, calcium dyshomeostasis and memory impairment induced by Aβ. Because Aβ toxicity is central to AD progression, the inhibition mediated by this new molecule might be useful as a therapeutic tool.

Amphiphilic polycations with quaternary ammonium groups show a strong tendency to porate lipid membranes. The polycations with a high capacity to open pores in biomembranes are the most cytotoxic to cells.