Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Abstract

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Fig. 1. Sequestration of Aβ-42 into the condensates inhibits fibril formation. (A)–(C) Confocal fluorescence microscopy (top panels) and brightfield microscopy images (bottom panels) of a solution of 10 μM Laf1-AK-Laf1 (A), Dbp1N-AK-Dbp1N (B) or Ddx4 (C) with (left panels) and without (right panels) 7 μM Aβ-42 labelled with the dye Atto-488 in 20 mM phosphate buffer at pH 8.0. (D) Measurements of Aβ-42 concentration in the continuous phase by monitoring the aggregation profile of the supernatant solution obtained after removal of the dispersed phase. 3 μM Aβ-42 solution was incubated in the absence (black curve) and presence of 0.5 μM Laf1-AK-Laf1 (blue curve) or 10 μM Laf1-AK-Laf1 (dark blue circles). From the measured half-time, the concentration of Aβ-42 in the supernatant (m_sup) is estimated from a known calibration curve determined from data reported in the literature. (E) Aggregation profiles of 2 μM Aβ-42 solution in the absence (black symbols) and presence (blue symbols) of 10 μM Laf1-AK-Laf1.

Fig. 2. Inhibition of Aβ-42 fibril formation in the presence of the condensates (A) time evolution of the ThT fluorescence intensity of samples incubated in glass bottom plates for 3 days: 4 μM Aβ-42 solution without (violet) and with 20 μM Laf1-AK-Laf1 (orange). The dispersed phase was re-suspended by mechanical agitation before analysis. (B) Residual Aβ-42 monomer measured by size exclusion chromatography. Values correspond to the areas under the monomer peak in the chromatograms measured for freshly purified monomeric 4 μM Aβ-42 solution before incubation (“Before”) and after incubation without (−LL) and with (+LL) 20 μM Laf1-AK-Laf1 (“After”). (C) and (D) Representative TEM images of fibrils formed in homogeneous solutions of 4 μM Aβ-42 in the absence of Laf1-AK-Laf1 after 3 days (C), and of a solution of 4 μM Aβ-42 incubated with 20 μM Laf1-AK-Laf1 for 3 days, showing lack of fibrils (D).

Fig. 3. Condensates inhibit Aβ-42 fibril formation in a dose dependent manner. (A) Aggregation profiles of 4 μM Aβ-42 solution in the absence and presence of 0.25 μM, 0.5 μM and 1 μM Laf1-AK-Laf1 (from left to right). Solid lines represent model simulations. (B) From the analysis of the aggregation profiles in (A) we estimated the concentration of Aβ-42 in the continuous phase (m_s) in the presence of increasing volume fractions of the dispersed phase (Φ_D). Solid line indicates the fit based on the overall mass balance. (C) Aggregation profiles of solutions containing 5.5 μM, 5 μM, 4 μM and 3 μM Aβ-42 at constant 0.5 μM Laf1-AK-Laf1. Solid lines indicate model simulations. (D) Concentration of Aβ-42 in the continuous phase estimated from the model simulations in (C) versus initial Aβ-42 concentrations (m⁰_Aβ). Solid line indicates fit based on the overall mass balance. (E) and (F) Confocal brightfield (left) and fluorescence (right) images of 2 μM Aβ-42 incubated with 5 μM Laf1-AK-Laf1 and 20 μM ThT after 30 min (E) and 150 min (F) of incubation. This experiment required a different multi-well plate compared to panels (A)–(C), leading to different aggregation kinetics (see Materials and methods).

Fig. 4. Condensates composed of different LCDs inhibit Aβ-42 fibril formation. (A) Aggregation profiles of 3.75 μM Aβ-42 solution in the absence (black) and presence of 1 μM, 2 μM, 5 μM and 10 μM Dpb1N-AK-Dpb1N (from left to right). (B) Aggregation profiles of 5 μM Aβ-42 solution in the absence (black) and presence of 1 μM, 5 μM, 10 μM and 20 μM (from left to right) LCD derived from Ddx4. In both panels solid lines represent model simulations that consider a decrease in the effective initial monomer concentration.

Fig. 5. Polymeric condensates preferentially exclude Aβ-42 and do not affect fibril formation. (A) and (B) Brightfield (left) and fluorescence (right) confocal images of 1 μM Aβ-42 labelled with Atto-488 incubated with coacervates formed by the zwitterionic polymer (A) and by the LCST polymer (B). (C) Aggregation profiles of 5 μM Aβ-42 in the absence (black) and presence of 20 μM zwitterionic polymer (orange) and 20 μM LCST polymer (green) at 27.5 °C.