scholarly journals Sequence determinants for the aggregation and cytotoxicity of proteins within condensates generated by liquid-liquid phase separation

2020 ◽  
Author(s):  
Michele Vendruscolo ◽  
Monika Fuxreiter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Human Disease ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Functional Protein ◽  
Mechanisms Of Toxicity ◽  
Different Types ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractThe transition between the native and amyloid states of proteins can proceed via a deposition pathway through oligomeric intermediates or via a condensation pathway through liquid droplet intermediates generated through liquid-liquid phase separation. The maturation of these droplet intermediates into ordered assemblies has been associated with human disease, including in particular amyotrophic lateral sclerosis (ALS), although the mechanisms of toxicity have not been yet clarified. Here we investigate the processes by which ALS-related mutations give rise to cytotoxicity along the condensation pathway. Based on the sequence-determinants of the different types of interactions stabilising the droplet and amyloid states, we accurately predict the levels of toxicity of about 50,000 deep mutagenesis variants of TDP-43 prion-like domain. We find that condensation is not typically initiated by structural ordering, but rather through non-specific interactions, and that the cytotoxicity of ALS-related TDP-43 mutations stems from promiscuous interactions within the droplet intermediates, rather than from the mature aggregates. These results provide insights into the mechanisms by which condensates convert into amyloids and their links with human disease.SignificanceProtein liquid-liquid phase separation underlies the formation of functional protein condensates, which upon dysregulation can mature into cytotoxic amyloid-containing aggregates. The sequence-based principles governing this pathway, and the mechanisms giving rise to cytotoxicity, however, are still not known in detail. Here, based on the different amino acid codes leading to the droplet and amyloid states, we show how one can predict the toxicity of the intermediate states along the condensation pathway. Our results highlight that this toxicity originates from the interaction promiscuity of amyloid-containing intermediates, in particular those with ALS-related mutations, rather than from the mature amyloid state. These results contribute to our understanding of the mechanisms through which TDP-43 mutations are linked to ALS.

scholarly journals Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 548
Author(s):  
Donya Pakravan ◽  
Emiel Michiels ◽  
Anna Bratek-Skicki ◽  
Mathias De Decker ◽  
Joris Van Lindt ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Hydrophobic Interactions ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
End Stage ◽  
Positive Effect ◽  
Self Aggregation ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

scholarly journals Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

2019 ◽  
Author(s):  
Soumik Ray ◽  
Nitu Singh ◽  
Satyaprakash Pandey ◽  
Rakesh Kumar ◽  
Laxmikant Gadhe ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Amyloid Formation ◽  
Liquid To Solid Transition ◽  
Phase Separation Behavior ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

SUMMARYα-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote α-Syn LLPS and its subsequent maturation. We further demonstrate α-Syn liquid droplet formation in cells, under oxidative stress. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

scholarly journals Role and therapeutic potential of liquid‒liquid phase separation in amyotrophic lateral sclerosis

2020 ◽  
Author(s):  
Donya Pakravan ◽  
Gabriele Orlando ◽  
Valérie Bercier ◽  
Ludo Van Den Bosch
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Phase Separation ◽  
Liquid Phase ◽  
Therapeutic Potential ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Familial Als ◽  
Als Patients ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

Abstract Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease selectively affecting motor neurons, leading to progressive paralysis. Although most cases are sporadic, ∼10% are familial. Similar proteins are found in aggregates in sporadic and familial ALS, and over the last decade, research has been focused on the underlying nature of this common pathology. Notably, TDP-43 inclusions are found in almost all ALS patients, while FUS inclusions have been reported in some familial ALS patients. Both TDP-43 and FUS possess ‘low-complexity domains’ (LCDs) and are considered as ‘intrinsically disordered proteins’ (IDPs), which form liquid droplets in vitro due to the weak interactions caused by the LCDs. Dysfunctional ‘liquid‒liquid phase separation’ (LLPS) emerged as a new mechanism linking ALS-related proteins to pathogenesis. Here, we review the current state of knowledge on ALS-related gene products associated with a proteinopathy and discuss their status as LLPS proteins. In addition, we highlight the therapeutic potential of targeting LLPS for treating ALS.

scholarly journals Regulation of TDP-43 phosphorylation in aging and disease

GeroScience ◽  
2021 ◽  
Author(s):  
Randall J. Eck ◽  
Brian C. Kraemer ◽  
Nicole F. Liachko
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Phase Separation ◽  
Liquid Phase ◽  
Frontotemporal Lobar Degeneration ◽  
Therapeutic Strategy ◽  
Liquid Phase Separation ◽  
Phosphatase Activities ◽  
Neurotoxic Effects ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractInsoluble inclusions of phosphorylated TDP-43 occur in disease-affected neurons of most patients with amyotrophic lateral sclerosis (ALS) and about half of patients with frontotemporal lobar degeneration (FTLD-TDP). Phosphorylated TDP-43 potentiates a number of neurotoxic effects including reduced liquid–liquid phase separation dynamicity, changes in splicing, cytoplasmic mislocalization, and aggregation. Accumulating evidence suggests a balance of kinase and phosphatase activities control TDP-43 phosphorylation. Dysregulation of these processes may lead to an increase in phosphorylated TDP-43, ultimately contributing to neurotoxicity and neurodegeneration in disease. Here we summarize the evolving understanding of major regulators of TDP-43 phosphorylation as well as downstream consequences of their activities. Interventions restoring kinase and phosphatase balance may be a generalizable therapeutic strategy for all TDP-43 proteinopathies including ALS and FTLD-TDP.

scholarly journals Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness

2021 ◽  
Author(s):  
Xin Jin ◽  
Ji-Eun Lee ◽  
Charley Schaefer ◽  
Xinwei Luo ◽  
Adam JM Wollman ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Quantitative Agreement ◽  
Liquid Phase Separation ◽  
Cell Processes ◽  
Bacterial Fitness ◽  
Membrane Bound ◽  
Multiple Species ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation is emerging as a crucial phenomenon in several fundamental cell processes. A range of eukaryotic systems exhibit liquid condensates. However, their function in bacteria, which in general lack membrane-bound compartments, remains less clear. Here, we used high-resolution optical microscopy to observe single bacterial aggresomes, nanostructured intracellular assemblies of proteins, to undercover their role in cell stress. We find that proteins inside aggresomes are mobile and undergo dynamic turnover, consistent with a liquid state. Our observations are in quantitative agreement with phase-separated liquid droplet formation driven by interacting proteins under thermal equilibrium that nucleate following diffusive collisions in the cytoplasm. We have discovered aggresomes in multiple species of bacteria, and show that these emergent, metastable liquid-structured protein assemblies increase bacterial fitness by enabling cells to tolerate environmental stresses.

scholarly journals Physical observables to determine the nature of membrane-less cellular sub-compartments

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mathias L Heltberg ◽  
Judith Miné-Hattab ◽  
Angela Taddei ◽  
Aleksandra M Walczak ◽  
Thierry Mora
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Site ◽  
Binding Sites ◽  
Spatial Organization ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Site Model ◽  
Multiple Binding ◽  
Liquid Liquid Phase Separation

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. We use our results to reject the multiple binding site model for Rad52 foci, and find a picture consistent with a liquid-liquid phase separation. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

scholarly journals Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

2019 ◽  
Author(s):  
Amandeep Girdhar ◽  
Vidhya Bharathi ◽  
Vikas Ramyagya Tiwari ◽  
Suman Abhishek ◽  
Usha Saraswat Mahawar ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Site ◽  
Liquid Phase Separation ◽  
Rna Molecules ◽  
Mechanism Of Inhibition ◽  
Rna Depletion ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractTDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc. Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43’s aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-432C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43’s aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43’s anti-aggregation effects with relevance to the ALS pathogenesis.

scholarly journals The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

2019 ◽  
Author(s):  
Stefan Reber ◽  
Helen Lindsay ◽  
Anny Devoy ◽  
Daniel Jutzi ◽  
Jonas Mechtersheimer ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Liquid Phase Separation ◽  
Fused In Sarcoma ◽  
Damage Repair ◽  
Interaction Partners ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

scholarly journals Liquid-liquid phase separation in secondary organic aerosol particles produced from α-pinene ozonolysis and α-pinene photo-oxidation with/without ammonia

2019 ◽  
Author(s):  
Suhan Ham ◽  
Zaeem Bin Babar ◽  
Jaebong Lee ◽  
Hojin Lim ◽  
Mijung Song
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Secondary Organic Aerosol ◽  
Cloud Condensation Nuclei ◽  
Aerosol Particles ◽  
Organic Aerosol ◽  
Liquid Phase Separation ◽  
Photo Oxidation ◽  
Different Types ◽  
Liquid Liquid Phase Separation

Abstract. Recently, liquid–liquid phase separation (LLPS) of secondary organic aerosol (SOA) particles free of inorganic salts has been intensively studied because of their importance on cloud condensation nuclei (CCN) properties. Herein, we investigated LLPS in four different types of SOA particles generated from α-pinene ozonolysis and α-pinene photo-oxidation in the absence and presence of NH3. LLPS was observed in SOA particles produced from α-pinene ozonolysis at ~ 95.8 % relative humidity (RH) and α-pinene ozonolysis with NH3 at ~ 95.4 % RH. However, LLPS was not observed in SOA particles produced from α-pinene photo-oxidation and α-pinene photo-oxidation with NH3. With datasets of average oxygen to carbon elemental ratio (O : C) for different types of SOA particles of this study and previous studies, LLPS occurred when the O : C ratio was less than ~ 0.44 and LLPS did not occur when the O : C ratio was greater than ~ 0.40. When LLPS was observed, the two liquid phases were present up to ~ 100 % RH. This result can help to predict more accurate results of CCN properties of organic aerosol particles.

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