Chemical probes for investigating protein liquid-liquid phase separation and aggregation

Protein liquid-liquid phase separation drives the dynamic assembly of membraneless organelles for fulfilling different physiological functions. Under diseased condition, protein may undergo liquid-to-solid condensation to form pathological amyloid aggregates closely associated with neurodegenerative diseases. Chemical probe serves as an important chemical tool not only for exploring the basic principle of the dynamic assembly of different protein condensates in vitro and in cell but also for clinical diagnosis and therapeutics of the related diseases. In this review, we first introduce chemical probes to image and regulate protein condensates. Then, we summarized three different categories of chemical probes including general amyloid dye, selective positron emission tomography tracer, and disaggregating binder, which feature distinct interaction pattern and activity upon binding to different pathological amyloid fibrillar aggregates. Next, we discuss the development of chemical probes for tracking protein amorphous aggregates in cells. Finally, we point out future direction in expanding the probes’ chemical space and applications.     

Segregative phase separation in agarose/whey protein systems induced by sequence-dependent trapping and change in pH

The structural properties and morphology of mixed gels made of aqueous preparations of agarose and whey protein were modified by changing thermal treatment and pH. The conformationally dissimilar polymers phase separated and this process was followed by small-deformation dynamic oscillation in shear, differential scanning calorimetry and environmental scanning electron microscopy. Experimental protocol encourages formation of a range of two-phase systems from continuous agarose matrices perforated by liquid-like whey protein inclusions to phase inverted preparations where a soft protein matrix suspends hard agarose-filler particles. These distinct morphologies have widely different mechanical moduli, which were followed by adapting a theoretical analysis (isostress-isostrain and Lewis-Nielsen blending laws) from the literature in synthetic block polymers and polyblends. Based on this framework of thought, reasonable predictions of the elastic moduli in the composite gels were made that led to patterns of solvent partition between the two polymeric networks. It was shown that proteins, in mixture with polysaccharide, exhibit favorable relative affinity (P-factor) for water molecules at a pH above their isoelectric point. This is an unexpected outcome that adds to the central finding of a single P value for the distribution of solvent between the continuous matrix and discontinuous inclusions of binary gels. It was thus proposed that phase continuity and solvent distribution in agarose/whey protein systems are under kinetic control that can be heavily governed by pH changes in the aqueous environment.     

Nucleic acid actions on abnormal protein aggregation,phase transitions and phase separation

Liquid–liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.     

Exploring protein aggregation and self-propagation using lattice models: phase diagram and kinetics

Many seemingly unrelated neurodegenerative disorders, such as amyloid and prion diseases, are associated with propagating fibrils whose structures are dramatically different from the native states of the corresponding monomers. This observation, along with the experimental demonstration that any protein can aggregate to form either fibrils or amorphous structures (inclusion bodies) under appropriate external conditions, suggest that there must be general principles that govern aggregation mechanisms. To probe generic aspects of prion-like behavior we use the model of Harrison, Chan, Prusiner, and Cohen. In this model, aggregation of a structure, that is conformationally distinct from the native state of the monomer, occurs by three parallel routes. Kinetic partitioning, which leads to parallel assembly pathways, occurs early in the aggregation process. In all pathways transient unfolding precedes polymerization and self-propagation. Chain polymerization is consistent with templated assembly, with the dimer being the minimal nucleus. The kinetic effciency of R(n-1) + G --> R(n) (R is the aggregation prone state and G is either U, the unfolded state, or N, the native state of the monomer) is increased when polymerization occurs in the presence of a "seed" (a dimer). These results support the seeded nucleated-polymerization model of fibril formation in amyloid peptides. To probe generic aspects of aggregation in two-state proteins, we use lattice models with side chains. The phase diagram in the (T,C) plane (T is the temperature and C is the polypeptide concentration) reveals a bewildering array of "phases" or structures. Explicit computations for dimers show that there are at least six phases including ordered structures and amorphous aggregates. In the ordered region of the phase diagram there are three distinct structures. We find ordered dimers (OD) in which each monomer is in the folded state and the interaction between the monomers occurs via a well-defined interface. In the domain-swapped structures a certain fraction of intrachain contacts are replaced by interchain contacts. In the parallel dimers the interface is stabilized by favorable intermolecular hydrophobic interactions. The kinetics of folding to OD shows that aggregation proceeds directly from U in a dynamically cooperative manner without populating partially structured intermediates. These results support the experimental observation that ordered aggregation in the two-state folders U1A and CI2 takes place from U. The contrasting aggregation processes in the two models suggest that there are several distinct mechanisms for polymerization that depend not only on the polypeptide sequence but also on external conditions (such as C, T, pH, and salt concentration).     

Tau protein liquid–liquid phase separation can initiate tau aggregation

The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.     

Emerging roles of O-glycosylation in regulating protein aggregation,phase separation,and functions

Protein O-glycosylation is widely identified in various proteins involved in diverse biological processes. Recent studies have demonstrated that O-glycosylation plays crucial and multifaceted roles in modulating protein amyloid aggregation and liquid–liquid phase separation (LLPS) under physiological conditions. Dysregulation of these processes is closely associated with human diseases such as neurodegenerative diseases (NDs) and cancers. In this review, we first summarize the distinct roles of O-glycosylation in regulating pathological aggregation of different amyloid proteins related to NDs and elaborate the underlying mechanisms of how O-glycosylation modulates protein aggregation kinetics, induces new aggregated structures, and mediates the pathogenesis of amyloid aggregates under diseased conditions. Furthermore, we introduce recent discoveries on O-GlcNAc-mediated regulation of synaptic LLPS and phase separation potency of low-complexity domain-enriched proteins. Finally, we identify challenges in future research and highlight the potential for developing new therapeutic strategies of NDs by targeting protein O-glycosylation.     

Thermodynamics and kinetics of phase separation of protein-RNA mixtures by a minimal model

Intracellular liquid-liquid phase separation enables the formation of biomolecular condensates, such as ribonucleoprotein granules, which play a crucial role in the spatiotemporal organization of biomolecules (e.g., proteins and RNAs). Here, we introduce a patchy-particle polymer model to investigate liquid-liquid phase separation of protein-RNA mixtures. We demonstrate that at low to moderate concentrations, RNA enhances the stability of RNA-binding protein condensates because it increases the molecular connectivity of the condensed-liquid phase. Importantly, we find that RNA can also accelerate the nucleation stage of phase separation. Additionally, we assess how the capacity of RNA to increase the stability of condensates is modulated by the relative protein-protein/protein-RNA binding strengths. We find that phase separation and multiphase organization of multicomponent condensates is favored when the RNA binds with higher affinity to the lower-valency proteins in the mixture than to the cognate higher-valency proteins. Collectively, our results shed light on the roles of RNA in ribonucleoprotein granule formation and the internal structuring of stress granules.     

Lipid phase separation induced by a hydrophobic protein in phosphatidylserine--phosphatidylcholine vesicles

Differential scanning calorimetry (DSC) was used to detect phase separation induced by hydrophobic myelin protein, lipophilin, in a mixture of phosphatidylserine (PS) and dipalmitoylphosphatidylcholine (DPPC). Preferential binding of PS to the boundary layer of lipophilin causes a decrease in the PS content of the remaining lamellar phase with a resultant shift in the phase-transition temperature to a higher temperature. The phase diagram for this mixture in the presence and absence of lipophilin is presented. From the phase diagram, it can be estimated that for an equimolar mixture of PS and DPPC, the boundary layer contains only PS, although for higher DPPC contents, some DPPC can also be found in the boundary layer. In the case where partial phase separation in induced in this mixture by Ca2+ alone, lipophilin increases the phase separation indicating that it also binds PS preferentially in the presence of Ca2+. Preferential binding of two other acidic lipids, phosphatidic acid and phosphatidyl-glycerol, to the boundary layer was also found, including a mixture where the acidic lipid was the higher melting component in the mixture.     

The kinetics of phase separation in asymmetric membranes

Phase separation in a model asymmetric membrane is studied using Monte Carlo techniques. The membrane comprises two species of particles, which mimic different lipids in lipid bilayers and separately possess either zero or non-zero spontaneous curvatures. We study the influence of phase separation on membrane shape and the influence of the coupling of composition and height dynamics on phase separation and domain growth, via both the degree of shape asymmetry and relative kinetic coefficients for height relaxation.     

A study of mass transfer kinetics in an enantiomeric separation system using a polymeric imprinted stationary phase

Chromatographic data pertaining to the enantioseparation of L- and D-phenylalanine anilide (PA) on a polymeric stationary phase imprinted with L-PA were studied from the viewpoints of phase equilibrium, mass transfer kinetics, and the thermodynamic properties of this enantiomeric separation system. The concentration dependence of the lumped mass transfer rate coefficient (k(m,L)) previously published was analyzed to obtain new information concerning the mass transfer characteristics in this chiral separation system. It was shown that intraparticle diffusion contributed much more to k(m,L) than adsorption/desorption. The positive concentration dependence of k(m,L) seemed to be interpreted by considering that of the surface diffusion coefficient, itself explained by the heterogeneous surface model. The characteristic features of the phase equilibrium, the mass transfer kinetics, and the thermodynamics of the enantiomeric separation system probably result from the adsorption energy distribution on the surface of the imprinted phase having an exponential decay.     

On the kinetics of phase separation in aqueous two-phase systems

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R, relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.     

Anaerobic membrane reactor with phase separation for the treatment of cheese whey

Two-phase anaerobic digestion of cheese whey was investigated in a system consisting of a stirred acidogenic reactor followed by a stirred methanogenic reactor, the latter being coupled to a membrane filtration system to enable removal of soluble effluent whilst retaining solids. The acidogenic reactor was operated at a hydraulic retention time (HRT) of one day, giving maximum acidification of 52.25% with up to 5 g/l volatile fatty acids, of which 63.7% was acetic acid and 24.7% was propionic acid. The methanogenic reactor received an organic load up to 19.78 g COD/ld, corresponding to a HRT of 4 days, at which 79% CODs and 83% BOD(5) removal efficiencies were obtained. Average removals of COD, BOD(5) and TSS in the two-phase anaerobic digestion process were 98.5%, 99% and 100%, respectively. The daily biogas production exceeded 10 times reactor volume and biogas methane content was greater than 70%.     

Liquid-liquid phase separation in hemoglobins: distinct aggregation mechanisms of the beta6 mutants

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (beta6 Lys) compared to HbS (beta6 Val) and HbA (beta6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the beta6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.     

Mechanism and kinetics of phase separation in a gelatin/maltodextrin mixture studied by small-angle light scattering

Phase separation mechanisms and kinetics were studied using small-angle light scattering in a gelatin/maltodextrin system where phase separation could be studied in both liquid and gelled states. Nucleation and growth or spinodal decomposition occurred, depending on the quench depth. The transition between the two mechanisms occurred relatively sharply. The different mechanisms were distinguishable by the different behavior of the scattering function even though a peak was observed in both cases. Particular differences were the different evolution of the peak intensity and position, the absence of dynamic scaling of the nucleation and growth scattering function, and the final coarsening exponent of 1/3 that was measured when spinodal decomposition occurred but not for nucleation and growth. Gelation severely reduced the coarsening rate and initially placed the phase compositions far from their equilibrium values. Despite the loss of molecular mobility caused by gelation, the gelled systems did continue to evolve, albeit much more slowly than in the liquid case. Multiple coarsening rates were observed for some of the gelled samples, which were ascribed to the gradual movement of these systems toward the equilibrium compositions.     

Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates

Soy-derived proteins (soy protein isolate, glycinin, and β-conglycinin) and bovine whey-derived proteins (whey protein isolate, -lactalbumin, β-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates obtained was studied as a function of pH. At neutral pH, all soy-derived protein hydrolysates, particularly those from glycinin, obtained by hydrolysis with subtilisin Carlsberg, chymotrypsin, bromelain, and papain showed a stronger aggregation compared to the non-hydrolyzed ones. This increase in aggregation was not observed upon hydrolysis by trypsin. None of the whey-derived protein hydrolysates exhibited an increase in aggregation at neutral pH. The high abundance of theoretical cleavage sites in the hydrophobic regions of glycinin probably explains the stronger exposure of hydrophobic groups than for the other proteins, which is suggested to be the driving force in the aggregate formation.     

Neurofilament protein aggregation in a cell line model system

Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.     

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.     

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.     

Effects of sugars on the cross-linking formation and phase separation of high-pressure induced gel of whey protein from bovine milk

The effects of sugars (xylose, arabinose, fucose, fructose, galactose, glucose, sorbitol, maltose, sucrose, and lactose; 0-20% w/v) on the properties of the pressure-induced gel from a whey protein isolate (20%, 800 MPa, 30 degrees C, 10 min) were studied. All the sugars decreased the hardness, breaking stress and water-holding capacity of the gel at the same concentration of 55.5 mM. Increasing the sugar content changed the microstructure of the gel from a honeycomb-like structure to a stranded structure, while the strand thickness was progressively reduced. These results suggest that sugars decreased the degree of intermolecular S-S bonding of proteins and non-covalent interaction, and restrained the phase separation during gelation under high pressure.     

Two-dimensional protein separation by the HPLC system with a monolithic column

A two-dimensional high-performance liquid chromatography (2D-HPLC) system for protein separation was developed using an ion-exchange column in the first dimension and a reversed-phase monolithic column in the second dimension. The system demonstrated efficient separation of proteins in comparison with conventional systems. For proteomic analysis, proteins extracted from the cell surface of the yeast were separated by 2D-HPLC and evaluated.