Liquid-Liquid Phase Separation and Its Mechanistic Role in Pathological Protein Aggregation

Abstract

Liquid-liquid phase separation (LLPS) of proteins underlies the formation of membrane-less organelles. While it has been recognized for some time that these organelles are of key importance for normal cellular functions, a growing number of recent observations indicate that LLPS may also play a role in disease. In particular, numerous proteins that form toxic aggregates in neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease, were found to be highly prone to phase separation, suggesting that there might be a strong link between LLPS and the pathogenic process in these disorders. This review aims to assess the molecular basis of this link through exploration of the intermolecular interactions that underlie LLPS and aggregation and the underlying mechanisms facilitating maturation of liquid droplets into more stable assemblies, including so-called labile fibrils, hydrogels, and pathological amyloids. Recent insights into the structural basis of labile fibrils and potential mechanisms by which these relatively unstable structures could transition into more stable pathogenic amyloids are also discussed. Finally, this review explores how the environment of liquid droplets could modulate protein aggregation by altering kinetics of protein self-association, affecting folding of protein monomers, or changing aggregation pathways.

Keywords: amyloid; liquid–liquid phase separation; neurodegenerative diseases; protein aggregation.

Figure 1.. Maturation of liquid droplets.

Changes in the material properties of liquid droplets can be monitored using fluorescence recovery after photobleaching, which typically involves pre-bleach, bleach, and post-bleach (or recovery) stages. Initiation of liquid droplet formation classically results in highly dynamic assemblies that exhibit rapid and almost complete recovery after photobleaching (though these properties may vary between proteins and with the addition of co-factors). Removal of the LLPS-inducing conditions (i.e., change in temperature, addition or dilution of salt) results in complete dissolution of droplets, indicating high reversibility. Freshly-prepared droplets grow in size and undergo fusion events over time. The term maturation describes a decrease in the extent of recovery after photobleaching with time until recovery is no longer observed. Such loss of dynamicity has been thought of as a gelation or aggregation phenomenon; however, the exact nature of such species is not always entirely clear. Additionally, this loss of dynamicity often coincides with a loss of reversibility when LLPS-promoting conditions are removed.

Figure 2.. Characteristic features of classical amyloids.

(A) Pathological amyloids are characterized by three defining features: fibrillar morphology, cross-β architecture, and tinctorial properties (staining by amyloid-specific dyes). Fibrillar morphology can be assessed using atomic force microscopy (shown) or transmission electron microscopy. The cross-β structure is defined by the ~4.7 Å distance between individual β-strands of the same sheet, while the distance between sheets is variable, often ~10 Å. Thioflavin-T (ThT, an amyloid-specific dye) exhibits elevated fluorescence upon binding to fibrils and therefore can be used to monitor assembly. Typically, monitoring the ThT fluorescence intensity over time yields a sigmoidal curve with a nucleation/lag phase and an elongation/exponential phase. (B) The nucleation phase involves formation of an elongation-competent nucleus, which typically is oligomeric but can also be a misfolded monomer. This nucleus has the capacity to initiate templated assembly and therefore fibril growth. Additional interactions between pre-formed fibrils can facilitate gelation, though this is not necessarily the case for all amyloids.

Figure 3.. Structures governing the stability of fibrils.

Atomic structures of microcrystals formed by short peptides can be classified as having extended β-strand, kinked β-strand, or non-cross-β architecture. For all cross-β structures, strands stack perpendicular to the fibril axis (arrow or point) via hydrogen bonds between backbone amides. (A) Peptides derived from Sup35 (left) and β-amyloid (right) form steric zippers with tight interdigitation of side chains and a dry interface between mating sheets. Interdigitation may involve polar (e.g., Asn in Sup35) or hydrophobic (e.g., Ile in β-amyloid) side chains. Fibrils formed by these classical amyloids are typically regarded as stable [101]. (B) Reversible amyloid cores (RACs) are another type of extended β-strand architecture observed for microcrystals of hnRNPA1- (left) and FUS-derived peptides (right). These structures do not contain very tight interdigitation of side chains and are more labile than classical amyloids. Microcrystals of hnRNPA1 peptides are loosely stabilized by a steric zipper with a polar sheet interface, but destabilized by the presence of negatively charged Asp side chains. These Asp side chains face away from the intersheet interface and stack along the fibril axis (referred to as a stacking-D). The labile structure with an extended β-strand architecture formed by the FUS-derived peptide contains exceptionally loose interdigitation and is weakly stabilized by hydrogen bonding between water and Tyr hydroxyl groups at the inter-sheet interface. (C) Another category of labile structures, LARKS, involves kinked β-strands that have been described for microcrystals formed by FUS, HNRNPA1, TDP-43, and nup98 peptides (FUS peptide is shown here). Kinks may be present at Gly or aromatic residues and enable close approach of peptide backbones at the intersheet interface without inter-digitation of side chains. (D) Microcrystals of the same FUS peptide shown in (C) may also form a labile non-cross-β structure. Consequently, this peptide sequence has been referred to both as a LARKS and RAC. In contrast to those structures containing β-strands, this non-cross-β structure is described as having a “coil with a sharp kink” at the central Gly40. The ordered-coil architecture is stabilized by hydrogen bonding between the Tyr38 and Ty41 in the same rung and between Tyr38 and Ser42 of neighboring segments. Cartoon depictions partially adapted from ref [92].

Figure 4.. Models depicting formation of reversible and irreversible fibrils within the context of LLPS.

(Left) Dynamic droplets undergo maturation with time. In some cases, this maturation leads to a reversible gel-like state that may contain labile fibrils. Upon prolonged incubation, more stable fibrillar aggregates are formed. (Right) Three models can describe the relationship between reversible and irreversible fibrils. On one extreme (Model 1), fibrillar structure (and therefore stability) may be predetermined by amino acid sequence. Thus, a protein that is rich in LARKS or RACs will be prone to only form reversible fibrils (i.e., hnRNPA1 or FUS), whereas a protein rich in steric zipper-forming sequences will only form irreversible fibrils (i.e., TDP-43). In a larger protein, the balance of these motifs could be a key regulator of stability that could also be modulated by pathogenic mutations. If multiple motifs are present, lability may be lost with time if a threshold number of RACs- or LARKS-based interactions is reached as more motifs sequentially associate. On the opposite extreme (Model 2), the amino acid sequence may not strongly regulate the type of fibril structure observed. In this latter scenario, the same sequence could form different types of RACs or LARKS (as is seen for the ³⁷SYSGYS⁴² FUS peptide) or even steric zipper. It is unlikely that both models 1 and 2 adequately describe all scenarios and some combination of both may be more realistic, at least in some cases. A natural sequelae of these two models is the possibility that, while a specific sequence may have a predisposition to form one type of structure (i.e., a labile LARKS or RACs structure), this structure may become more stable with time and might even transition entirely to a steric zipper (Model 3). Post-translational modifications (PTMs) could also regulate such a transition. Cartoon depictions partially adapted from ref [102].