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

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.

Download Full-text

Liquid-liquid phase separation of the intrinsically disordered AB region of hRXRγ is driven by hydrophobic interactions

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2021.05.035 ◽

2021 ◽

Author(s):

Katarzyna Sołtys ◽

Krzysztof Wycisk ◽

Andrzej Ożyhar

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Hydrophobic Interactions ◽

Liquid Phase Separation ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

Download Full-text

Exploiting mammalian low-complexity domains for liquid-liquid phase separation–driven underwater adhesive coatings

Science Advances ◽

10.1126/sciadv.aax3155 ◽

2019 ◽

Vol 5 (8) ◽

pp. eaax3155 ◽

Cited By ~ 10

Author(s):

Mengkui Cui ◽

Xinyu Wang ◽

Bolin An ◽

Chen Zhang ◽

Xinrui Gui ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Soft Materials ◽

Low Complexity ◽

Liquid Phase Separation ◽

Biologically Inspired ◽

Underwater Adhesion ◽

Assembly Mechanism ◽

Adhesion Performance ◽

Liquid Liquid Phase Separation

Many biological materials form via liquid-liquid phase separation (LLPS), followed by maturation into a solid-like state. Here, using a biologically inspired assembly mechanism designed to recapitulate these sequential assemblies, we develop ultrastrong underwater adhesives made from engineered proteins containing mammalian low-complexity (LC) domains. We show that LC domain–mediated LLPS and maturation substantially promotes the wetting, adsorption, priming, and formation of dense, uniform amyloid nanofiber coatings on diverse surfaces (e.g., Teflon), and even penetrating difficult-to-access locations such as the interiors of microfluidic devices. Notably, these coatings can be deposited on substrates over a broad range of pH values (3 to 11) and salt concentrations (up to 1 M NaCl) and exhibit strong underwater adhesion performance. Beyond demonstrating the utility of mammalian LC domains for driving LLPS in soft materials applications, our study illustrates a powerful example of how combining LLPS with subsequent maturation steps can be harnessed for engineering protein-based materials.

Download Full-text

Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

10.1101/2021.10.08.463684 ◽

2021 ◽

Author(s):

Josep Rizo ◽

Carlos M. Roggero ◽

Victoria Esser ◽

Lingling Duan ◽

Allyson M. Rice ◽

...

Keyword(s):

Prostate Cancer ◽

Phase Separation ◽

Androgen Receptor ◽

Liquid Phase ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Low Complexity ◽

Liquid Phase Separation ◽

Liquid Liquid Phase Separation

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails dramatic epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC.

Download Full-text

The Role of Methionine Residues in the Regulation of Liquid-Liquid Phase Separation

Biomolecules ◽

10.3390/biom11081248 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1248

Author(s):

Juan Carlos Aledo

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Low Complexity ◽

Liquid Phase Separation ◽

Modular Architecture ◽

Residue Interaction ◽

Methionine Residues ◽

Molecular Forces ◽

Liquid Liquid Phase Separation

Membraneless organelles are non-stoichiometric supramolecular structures in the micron scale. These structures can be quickly assembled/disassembled in a regulated fashion in response to specific stimuli. Membraneless organelles contribute to the spatiotemporal compartmentalization of the cell, and they are involved in diverse cellular processes often, but not exclusively, related to RNA metabolism. Liquid-liquid phase separation, a reversible event involving demixing into two distinct liquid phases, provides a physical framework to gain insights concerning the molecular forces underlying the process and how they can be tuned according to the cellular needs. Proteins able to undergo phase separation usually present a modular architecture, which favors a multivalency-driven demixing. We discuss the role of low complexity regions in establishing networks of intra- and intermolecular interactions that collectively control the phase regime. Post-translational modifications of the residues present in these domains provide a convenient strategy to reshape the residue–residue interaction networks that determine the dynamics of phase separation. Focus will be placed on those proteins with low complexity domains exhibiting a biased composition towards the amino acid methionine and the prominent role that reversible methionine sulfoxidation plays in the assembly/disassembly of biomolecular condensates.

Download Full-text

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

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa049 ◽

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.

Download Full-text

The C-terminal low-complexity domain involved in liquid–liquid phase separation is required for BRD4 function in vivo

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz037 ◽

2019 ◽

Vol 11 (9) ◽

pp. 807-809

Author(s):

Chenlu Wang ◽

Erhao Zhang ◽

Fan Wu ◽

Yufeng Sun ◽

Yingcheng Wu ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Low Complexity ◽

Liquid Phase Separation ◽

Liquid Liquid Phase Separation

Download Full-text

Regulation of TDP-43 phosphorylation in aging and disease

GeroScience ◽

10.1007/s11357-021-00383-5 ◽

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.

Download Full-text

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

10.1101/2021.03.05.434000 ◽

2021 ◽

Author(s):

Einav Tayeb-Fligelman ◽

Xinyi Cheng ◽

Christen Tai ◽

Jeannette T. Bowler ◽

Sarah Griner ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Low Complexity ◽

Fibril Formation ◽

Liquid Phase Separation ◽

Amyloid Formation ◽

Rna Packaging ◽

Atomic Structures ◽

Amyloid Fibril Formation ◽

Liquid Liquid Phase Separation

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19.

Download Full-text