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. 2021 Sep 1;12(17):3189-3202.
doi: 10.1021/acschemneuro.1c00327. Epub 2021 Aug 12.

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

Affiliations

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 et al. ACS Chem Neurosci. .

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.

Keywords: Alzheimer’s disease; Parkinson’s disease; aminosterols; neurodegeneration; protein misfolding; squalamine.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M.Z. is one of the inventors in a patent for the use of trodusquemine in the treatment of Parkinson's disease. M.V. is a director of Wren Therapeutics Ltd, which is independently pursuing inhibitors of protein aggregation. The remaining authors declare no competing interests.

Figures

Figure 1
Figure 1
Binding of OAs/OBs/native HypF-N to LUVs. (A,B) Stern–Volmer plots reporting the ratio of fluorescence of TMA-DPH (A) and DPH (B) in 0.3 mg/mL LUVs in the absence (F0) or presence (F) of various concentrations (monomer equivalents) of OAs (red circles), OBs (blue triangles), and native HypF-N (green squares). The straight lines through the data points represent the best fits to eq 4. (C) Binding plots reporting the fluorescence at 512 nm of 20 μM BODIPY-FL-labeled OAs (red circles), OBs (blue triangles), and native HypF-N (green squares) versus LUV concentration reported in mg/mL units (bottom x axis) or mM units (top x axis). The lines through the data points represent the best fits to eq 6. (D,E) Bar plots reporting the KSV values obtained from TMA-DPH (D) and DPH (E) fluorescence quenching using eq 4. (F) Bar plots reporting the KD values from binding using eq 6. Experimental errors represent SEM of 2–5 experiments. The symbols * and *** refer to p values of <0.1 and <0.001, respectively, relative to KSV values of the native protein (D,E) and relative to the KD value of OAs (F).
Figure 2
Figure 2
Far-UV CD and intrinsic fluorescence spectra of OAs, OBs, and native HypF-N. (A–C) Far UV CD spectra of OAs (A), OBs (B), and native HypF-N (C) in the presence of increasing concentrations of LUVs. Spectra were blank-subtracted and normalized using eq 1. (D–F) Intrinsic tryptophan fluorescence emission spectra of OAs (D), OBs (E), and native HypF-N (F) in the presence of increasing concentrations of LUVs.
Figure 3
Figure 3
Intraoligomer FRET between OAs and OBs LUVs. (A,B) Fluorescence emission spectra of OAs (A) and OBs (B) formed by 18D_10 (green), 10_34A (red), and 18D+34A (blue), obtained in the presence of increasing concentrations of LUVs (0.12, 0.3, 0.5, and 0.7 mg/mL). (C) FRET E values of OAs (gray) and OBs (black) in the presence of increasing concentrations of LUVs, determined using eq 2. Experimental errors are SD.
Figure 4
Figure 4
FRET between OAs labeleld with donor (D) and the various lipids labeled with acceptor (A) contained in LUVs. (A) Fluorescence emission spectra of OA-D+Lipid-A (blue), OA-D (green), and Lipid-A (red). (B) FRET E values of the indicated FRET pairs examined, obtained using eq 3. Experimental errors represent SEM of 5 experiments.
Figure 5
Figure 5
Interaction of OAs with LUVs with and without trodusquemine. (A,B) Stern–Volmer plots reporting the ratio of fluorescence of TMA-DPH (A) and DPH (B) in the absence (F0) or presence (F) of various concentrations (monomer equivalents) of OAs, in the absence (red circles) and presence (black diamonds) of 5 μM trodusquemine (TRO) in 0.3 mg/mL LUVs. The straight lines through the data points represent the best fits to eq 4 (red line) and eq 5 (black line). Experimental errors represent SEM of 2–5 experiments. (C) Binding plots reporting the fluorescence at 512 nm of OAs in the absence (red circles) and presence (black diamonds) of TRO in LUVs, versus LUV concentration. The lines through the data points represent the best fits to eq 6. (D,E) Bar plots reporting the KSV values obtained from TMA-DPH (D) and DPH (E) fluorescence quenching in the absence (red) and presence (black) of 5 μM trodusquemine. (F) Bar plots reporting the KD values obtained from the binding experiments of OAs in the absence (red) and presence (black) of 5 μM trodusquemine. Experimental errors represent SEM of 2–5 experiments. The symbols *** refer to p values of <0.001 relative to KSV (D,E) and KD (F) values of OAs in the absence of trodusquemine.
Figure 6
Figure 6
Binding of OAs to LUVs with various lipid compositions. (A,B) Stern–Volmer plots reporting the ratio of fluorescence of TMA-DPH in the absence (F0) or presence (F) of various concentrations (monomer equivalents) of OAs, in the absence (various shades of red circles), and in the presence (various shades of gray diamonds) of 5 μM trodusquemine (TRO) in 0.3 mg/mL LUVs containing different percentage of GM1 (A) and CHOL (B). The straight lines through the data points represent the best fits to eq 4 (various shades of red lines) and eq 5 (various shades of gray lines). (C,D) Bar plots reporting the KSV values obtained from TMA-DPH fluorescence quenching in LUVs containing different percentages of GM1 (C) and CHOL (D) in the absence (various shades of red) and in the presence (various shades of gray) of 5 μM trodusquemine. Experimental errors represent SEM of 2–5 experiments. The symbols ** and *** refer to p values of <0.01 and <0.001, respectively, relative to KSV values of OAs without GM1 (C) and without CHOL (D) in the absence of trodusquemine; $ and $$$ refer to p values of <0.05 and <0.001, respectively, relative to KSV values of OAs without GM1 (C) and CHOL (D) in the presence of trodusquemine.
Figure 7
Figure 7
Summary of the results obtained in this work about the affinity of OAs and OBs for LUV lipid membranes. (A–C) Schematic representation of the affinity of the native protein (green, no affinity) (A), OBs (blue, low affinity) (B), and OAs (red, high affinity) (C) for the LUV lipid membrane. (D–F) Change of the OAs affinity due to the addition of trodusquemine (decreased affinity) (D), increase of GM1 concentration (increased affinity) (E), and increase of CHOL concentration (slightly decreased affinity) (F) in LUVs.

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