Quantitative Measurement of the Affinity of Toxic and Nontoxic Misfolded Protein Oligomers for Lipid Bilayers and of its Modulation by Lipid Composition and Trodusquemine

Abstract

Many neurodegenerative diseases are associated with the self-assembly of peptides and proteins into fibrillar aggregates. Soluble misfolded oligomers formed during the aggregation process, or released by mature fibrils, play a relevant role in neurodegenerative processes through their interactions with neuronal membranes. However, the determinants of the cytotoxicity of these oligomers are still unclear. Here we used liposomes and toxic and nontoxic oligomers formed by the same protein to measure quantitatively the affinity of the two oligomeric species for lipid membranes. To this aim, we quantified the perturbation to the lipid membranes caused by the two oligomers by using the fluorescence quenching of two probes embedded in the polar and apolar regions of the lipid membranes and a well-defined protein−oligomer binding assay using fluorescently labeled oligomers to determine the Stern−Volmer and dissociation constants, respectively. With both approaches, we found that the toxic oligomers have a membrane affinity 20−25 times higher than that of nontoxic oligomers. Circular dichroism, intrinsic fluorescence, and FRET indicated that neither oligomer type changes its structure upon membrane interaction. Using liposomes enriched with trodusquemine, a potential small molecule drug known to penetrate lipid membranes and make them refractory to toxic oligomers, we found that the membrane affinity of the oligomers was remarkably lower. At protective concentrations of the small molecule, the binding of the oligomers to the lipid membranes was fully prevented. Furthermore, the affinity of the toxic oligomers for the lipid membranes was found to increase and slightly decrease with GM1 ganglioside and cholesterol content, respectively, indicating that physicochemical properties of lipid membranes modulate their affinity for misfolded oligomeric species.

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design

“…The formation of amyloid plaques deposited on the extracellular membranes of neurons is one of the primary features of Alzheimer's disease [716]. This phenomenon is affected by specific lipids including cholesterol, sphingomyelin, and gangliosides [717,718]: lipid types typically present in the outer leaflet of the cell membrane. Amyloid fibers at a membrane surface have thus been frequently studied using MD simulations, e.g., [719][720][721][722][723][724][725][726][727][728][729][730], however, a surprisingly small fraction of this work has been performed in the context of drug design [731,732].…”

From Marine Metabolites to the Drugs of the Future: Squalamine, Trodusquemine, Their Steroid and Triterpene Analogues

“…Recent experiments confirmed that squalamine attenuates the toxicity of αS and amyloid-beta (Aβ) by altering their aggregation and displacing them from cell membranes [149]. Similar properties were later shown for trodusquemine and αS, amyloid-beta (Aβ), and HypF-N oligomers [118,[150][151][152]. Squalamine effectively restores disordered colonic motility by restoring excitability of the enteric nervous system in a mouse model [15] and reduced toxicity of αS in a C. elegans model of Parkinson's disease [153].…”

“…Neuroprotective In vitro: alpha-synuclein, amyloid-beta [148,149]; in animal models of Parkinson's disease [15,153]; phase 2 clinical trilas of squalamine phosphate for Parkinson's disease [22,158] In vitro: alpha-synuclein, amyloid-beta, HypF-N [119,[152][153][154][155]157] In animal models of Parkinson's and Alzheimer's diseases (presumably PTP1B is also involved) [157,[159][160][161]…”

A Brain-Permeable Aminosterol Regulates Cell Membranes to Mitigate the Toxicity of Diverse Pore-Forming Agents

“…Schematic for the mechanism of action by which the folded toxins melittin (A) and α-hemolysin (B) disrupt and create pores in the cell membranes. The illustration shows how claramine (green) incorporates into the cell membrane to prevent the pore-forming toxins from docking, analogous to the case for protein misfolded oligomers observed in neurodegenerative diseases. ,,,, The graphic was generated from the knowledge that aminosterols localize within the hydrophilic region of the lipid bilayer and extend to the interface between the hydrophilic and hydrophobic regions with a well-defined oblique angle (about 55°) for the major axis of the molecule with respect to the normal to the bilayer plane and with superficial positioning of its positively charged spermine tail . As a result, the membrane becomes less negatively charged, and it causes a redistribution of cholesterol and ganglioside GM1 molecules and acquires resistance to indentation …”

“…As a result, the membrane becomes less negatively charged, protected against oligomer embedding, and it causes a redistribution of cholesterol and ganglioside GM1 molecules that are known to protect cells from oligomer toxicity . Given the observations of a conserved mechanism of action of the aminosterols against aggregates, including multiple types of protein misfolded oligomers, − we hypothesized that they could act as a regulator of the cell membrane to suppress the toxicity of a wide range of different threat agents.…”