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. 2019 Feb 22;4(2):4029-4039.
doi: 10.1021/acsomega.8b03590. eCollection 2019 Feb 28.

Reducing the Amyloidogenicity of Functional Amyloid Protein FapC Increases Its Ability To Inhibit α-Synuclein Fibrillation

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Free PMC article

Reducing the Amyloidogenicity of Functional Amyloid Protein FapC Increases Its Ability To Inhibit α-Synuclein Fibrillation

Line Friis Bakmann Christensen et al. ACS Omega. .
Free PMC article

Abstract

Functional amyloid (FA) proteins have evolved to assemble into fibrils with a characteristic cross-β structure, which stabilizes biofilms and contributes to bacterial virulence. Some of the most studied bacterial FAs are the curli protein CsgA, expressed in a wide range of bacteria, and FapC, produced mainly by members of the Pseudomonas genus. Though unrelated, both CsgA and FapC contain imperfect repeats believed to drive the formation of amyloid fibrils. While much is known about CsgA biogenesis and fibrillation, the mechanism of FapC fibrillation remains less explored. Here, we show that removing the three imperfect repeats of FapC (FapC ΔR1R2R3) slows down the fibrillation but does not prevent it. The increased lag phase seen for FapC ΔR1R2R3 allows for disulfide bond formation, which further delays fibrillation. Remarkably, these disulfide-bonded species of FapC ΔR1R2R3 also significantly delay the fibrillation of human α-synuclein, a key protein in Parkinson's disease pathology. This attenuation of α-synuclein fibrillation was not seen for the reduced form of FapC ΔR1R2R3. The results presented here shed light on the FapC fibrillation mechanism and emphasize how unrelated fibrillation systems may share such common fibril formation mechanisms, allowing inhibitors of one fibrillating protein to affect a completely different protein.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Both FapC and FapC ΔR1R2R3 form ThT-positive amyloid fibrils. (A) Fibrillation followed with ThT for 1 mg/mL FapC (=42 μM) (black) and 1 mg/mL FapC ΔR1R2R3 (=72 μM) (orange). After fibrillation, the fibrils were investigated with (B) FTIR and (C) TEM. The scale bar is 100 nm.
Figure 2
Figure 2
Monomers of FapC and FapC ΔR1R2R3 both preferentially recognize their own fibrils and recognize different species of α-SN. Dot blot of labelled FapC (left) or labelled FapC ΔR1R2R3 (right) binding to 12.5−2000 ng of (A) immobilized monomers (M) or fibrils (F) of either FapC or FapC ΔR1R2R3 and decreasing concentrations of (B) immobilized FapC/FapC ΔR1R2R3 monomers or (C) immobilized α-SN monomers, oligomers (O), and fibrils.
Figure 3
Figure 3
Inhibition of α-SN fibrillation by FapC ΔR1R2R3. (A) Fibrillation of 1 mg/mL α-SN (69 μM) in the presence of monomeric FapC ΔR1R2R3 at concentrations ranging from 0.004 mg/mL (0.3 μM) to 1 mg/mL (72 μM). Lag time of α-SN fibrillation as a function of (B) FapC ΔR1R2R3 or (C) FapC concentration. Data using monomeric FapC or FapC ΔR1R2R3 are from three individual experiments I–III (each in triplicate). The average from these three experiments is shown with a black line. Results from experiments using fibrils of FapC or FapC ΔR1R2R3 instead of monomeric protein are shown in green and indicate no significant effect.
Figure 4
Figure 4
FapC ΔR1R2R3 forms small, mixed oligomers with α-SN. α-SN (4 mg/mL, 275 μM) and either (A) 2 mg/mL FapC ΔR1R2R3 (145 μM) or (B) 2 mg/mL FapC (85 μM) were run on SEC before (no shaking) and after shaking (4.25 h, 37 °C, 700 rpm). α-SN was run either alone or in a α-SN/FapC ΔR1R2R3 ratio of 1:0.5. Notice the difference in the UV280 nm of the zoomed graphs. Numbers represent the precise elution volumes (in mL) obtained from the Gaussian fitting to the elution profiles. (C) From the SEC run with 1:0.5 α-SN/FapC ΔR1R2R3 after shaking, fractions 1–4 (zoom) were collected. (D) All four fractions were pooled, up-concentrated and immobilized on nitrocellulose membranes together with duplicates of 1 μg α-SN, 1 μg freshly desalted FapC ΔR1R2R3, and 1× PBS and investigated with antibodies against either α-SN (left) or FapC (right).
Figure 5
Figure 5
Inhibitory effect of FapC ΔR1R2R3 on α-SN fibrillation is lost under reducing conditions. (A) Fibrillation of 1 mg/mL α-SN (69 μM) in the presence of FapC ΔR1R2R3 concentrations ranging from 0.004 mg/mL (0.3 μM) to 1 mg/mL (72 μM) in the presence of DTT. (B) Effect of DTT on the fibrillation kinetics of FapC ΔR1R2R3 and (inset) FapC.
Figure 6
Figure 6
Proposed mechanism for inhibition of α-SN fibrillation by FapC ΔR1R2R3. For both α-SN (blue) and FapC ΔR1R2R3 (orange), monomers (spheres) can form oligomers that can be elongated further to mature fibrils. Because of its long lag phase, FapC ΔR1R2R3 will start forming dimers, trimers, and oligomers with disulfide bond linkages (yellow stars), and these species retard fibrillation of FapC ΔR1R2R3 itself as well as α-SN.

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