One of the hallmarks of Alzheimer’s disease and several other neurodegenerative disorders is the aggregation of tau protein into fibrillar structures. Building on recent reports that tau readily ...undergoes liquid–liquid phase separation (LLPS), here we explored the relationship between disease-related mutations, LLPS, and tau fibrillation. Our data demonstrate that, in contrast to previous suggestions, pathogenic mutations within the pseudorepeat region do not affect tau441’s propensity to form liquid droplets. LLPS does, however, greatly accelerate formation of fibrillar aggregates, and this effect is especially dramatic for tau441 variants with disease-related mutations. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of fibrillation in mixtures containing tau isoforms with different aggregation propensities. This regulation results from unique properties of proteins under LLPS conditions, where total concentration of all tau variants in the condensed phase is constant. Therefore, the presence of increasing proportions of the slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propensity, reducing the rate of its fibrillation. This regulatory mechanism may be of direct relevance to phenotypic variability of tauopathies, as the ratios of fast and slowly aggregating tau isoforms in brain varies substantially in different diseases.
Recent studies have indicated that tau, a protein involved in Alzheimer's disease and other neurodegenerative disorders, has a propensity to undergo liquid–liquid phase separation (LLPS). However, ...the mechanism of this process remains unknown. Here, we demonstrate that tau LLPS is largely driven by intermolecular electrostatic interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions, whereas hydrophobic interactions play a surprisingly small role. Furthermore, our results reveal that, in contrast to previous suggestions, phosphorylation is not required for tau LLPS. These findings provide a foundation for understanding the mechanism by which phosphorylation and other posttranslational modifications could modulate tau LLPS in the context of specific physiological functions as well as pathological interactions.
Aggregation of the microtubule-associated protein tau plays a major role in Alzheimer’s disease and several other neurodegenerative disorders. An exciting recent development is the finding that, akin ...to some other proteins associated with neurodegenerative disease, tau has a high propensity to condensate via the mechanism of liquid–liquid phase separation (LLPS). Here, we discuss the evidence for tau LLPS in vitro, the molecular mechanisms of this reaction, and the role of post-translational modifications and pathogenic mutations in tau phase separation. We also discuss recent studies on tau LLPS in cells and the insights these studies provide regarding the link between LLPS and neurodegeneration in tauopathies.
Tau alone or in the presence of RNA has a high propensity to undergo liquid–liquid phase separation (LLPS), forming liquid droplets.LLPS of full-length tau under physiologically relevant buffer conditions is largely driven by intermolecular electrostatic interactions between oppositely charged protein regions or between tau and RNA.Phosphorylation and other post-translational modifications appear to play a regulatory role in tau LLPS.LLPS has a major impact on tau aggregation into amyloid fibrils. It also leads to unique regulatory mechanisms of fibrillation when multiple tau isoforms are present within the condensates.Tau can undergo LLPS in neurons and this may contribute to normal biological functions of the protein as well as to tau pathology in neurodegenerative diseases.
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.
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•RNA-binding proteins undergo liquid phase separation and aggregate in disease.•Liquid droplets formed by these proteins mature with time.•These proteins commonly form labile fibrils, distinct from classical amyloids.•Fibril stability is based on specific structural characteristics.•Phase separation can modulate protein aggregation by several mechanisms.
Amyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the ...low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.
Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo ...liquid–liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders.
Pathological aggregation of the transactive response DNA-binding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic ...traumatic encephalopathy, and Alzheimer's disease. TDP-43 aggregation appears to be largely driven by its low-complexity domain (LCD), which also has a high propensity to undergo liquid–liquid phase separation (LLPS). However, the mechanism of TDP-43 LCD pathological aggregation and, most importantly, the relationship between the aggregation process and LLPS remains largely unknown. Here, we show that amyloid formation by the LCD is controlled by electrostatic repulsion. We also demonstrate that the liquid droplet environment strongly accelerates LCD fibrillation and that its aggregation under LLPS conditions involves several distinct events, culminating in rapid assembly of fibrillar aggregates that emanate from within mature liquid droplets. These combined results strongly suggest that LLPS may play a major role in pathological TDP-43 aggregation, contributing to pathogenesis in neurodegenerative diseases.
Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also ...facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.
Aggregation of tau is one of the major pathogenic events in Alzheimer’s disease and several other neurodegenerative disorders. Recent reports demonstrated that tau can condense into liquid droplets ...that undergo time-dependent transition to a solid-like state, suggesting that liquid condensates may be on the pathway to pathological aggregation of tau. While hyperphosphorylation is a key feature of tau isolated from brains of patients with Alzheimer’s disease and other tauopathies, the mechanistic role of phosphorylation in tau liquid-liquid phase separation (LLPS) remains largely unexplored. In an attempt to bridge this gap, here we performed systematic studies by introducing phosphomimetic substitutions of Ser/Thr residues with negatively charged Asp/Glu residues in different regions of the protein. Our data indicate that the phosphorylation patterns that increase the polarization of charge distribution in full-length tau (tau441) promote protein LLPS, whereas those that decrease charge polarization have an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive intermolecular electrostatic interactions between the oppositely charged domains. We also show that the phosphomimetic tau variants with low intrinsic propensity for LLPS can be efficiently recruited to droplets formed by the variants with high LLPS propensity. Furthermore, the present data demonstrate that phosphomimetic substitutions have a major effect on time-dependent material properties of tau droplets, generally slowing down their aging. The latter effect is most dramatic for the tau variant with substitutions within the repeat domain, which correlates with the decreased fibrillation rate of this variant.
Recombinant C-terminally truncated prion protein PrP23-144 (which corresponds to the Y145Stop PrP variant associated with a Gerstmann–Sträussler–Scheinker-like prion disease) spontaneously forms ...amyloid fibrils with a parallel in-register β-sheet architecture and β-sheet core mapping to residues ∼112–139. Here we report that mice (both tga20 and wild type) inoculated with a murine (moPrP23-144) version of these fibrils develop clinical prion disease with a 100% attack rate. Remarkably, even though fibrils in the inoculum lack the entire C-terminal domain of PrP, brains of clinically sick mice accumulate longer proteinase K-resistant (PrPres) fragments of ∼17–32 kDa, similar to those observed in classical scrapie strains. Shorter, Gerstmann–Sträussler–Scheinker-like PrPres fragments are also present. The evidence that moPrP23-144 amyloid fibrils generated in the absence of any cofactors are bona fide prions provides a strong support for the protein-only hypothesis of prion diseases in its pure form, arguing against the notion that nonproteinaceous cofactors are obligatory structural components of all infectious prions. Furthermore, our finding that a relatively short β-sheet core of PrP23-144 fibrils (residues ∼112–139) with a parallel in-register organization of β-strands is capable of seeding the conversion of full-length prion protein to the infectious form has important implications for the ongoing debate regarding structural aspects of prion protein conversion and molecular architecture of mammalian prions.
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