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

Silvia Errico,Claudio Canale,Michele Vendruscolo,Denise Barbut,Hassan Ramshini,Reinier Oropesa-Nuñez,Claudia Capitini,Michael Zasloff,Martino Calamai,Martina Spaziano,Fabrizio Chiti

doi:10.1021/acschemneuro.1c00327

Silvia Errico, Claudio Canale + Show 9 more

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https://doi.org/10.1021/acschemneuro.1c00327

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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.

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Many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Creutzfeld-Jacob disease (CJD), Huntington disease (HD), frontotemporal dementia (FTD), familial amyloid polyneuropathy (FAP), and many others are associated with the self-assembly of specific peptides or proteins into misfolded fibrillar aggregates known as amyloid fibrils.[1,2]
We reveal quantitatively the effects of trodusquemine, a promising and previously studied small molecule that binds to the membrane, on the OA−membrane affinity and on the mechanism of displacement of these toxic oligomers from the bilayer and, again in a quantitative manner, how the lipid composition of large unilamellar vesicles (LUVs) can influence the affinity of toxic oligomers for the membranes
The images show that OAs bind to the gel-phase domains (Lβ or So) and to the liquiddisordered phase (Lα or Ld) of the supported lipid bilayers (SLBs) with 1% GM1, whereas only few OBs were found to be bound to them (Figure S1), in agreement with previous results obtained with 5% GM1 as the only difference in LUV composition relative to our LUV preparations.[30]

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Introduction

Many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Creutzfeld-Jacob disease (CJD), Huntington disease (HD), frontotemporal dementia (FTD), familial amyloid polyneuropathy (FAP), and many others are associated with the self-assembly of specific peptides or proteins into misfolded fibrillar aggregates known as amyloid fibrils.[1,2] The formation of amyloid fibrils is a generic characteristic of proteins, and it is found for a large number of systems that are not associated with disease.[3−5]Multiple lines of evidence suggest that small soluble oligomers formed in the process of amyloid fibril formation or released by mature fibrils are important players of the neurotoxicity associated with protein aggregation.[1,6−10] These species have an ability to bind to and destabilize biological membranes, inducing an entry of Ca2+ from the extracellular space into the cytosol. 25-fold, relative to OAs. under the conditions used here, we have quantified the binding affinity of toxic OAs and nontoxic OBs of a sample protein for the bilayer of lipid vesicles (LUVs) by measuring the KSV values of fluorescence quenching of membraneembedded TMA-DPH and DPH caused the oligomers (0.3 mg/mL LUVs or 384 μM total lipids) and the oligomermembrane KD values (20 μM protein in monomer equivalents).

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