scholarly journals Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

2021 ◽  
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation ◽  
In Cells ◽  
Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

scholarly journals Integration of Data from Liquid–Liquid Phase Separation Databases Highlights Concentration and Dosage Sensitivity of LLPS Drivers

2021 ◽  
Vol 22 (6) ◽  
pp. 3017
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Systematic Difference ◽  
Liquid Phase Separation ◽  
In Vivo Studies ◽  
Supporting Evidence ◽  
Condensate Formation ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals Liquid–Liquid Phase Separation in Crowded Environments

2020 ◽  
Vol 21 (16) ◽  
pp. 5908 ◽  
Author(s):  
Alain A. M. André ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Macromolecular Crowding ◽  
Liquid Phase Separation ◽  
The Impact ◽  
Crowded Environments ◽  
Liquid Liquid Phase Separation ◽  
In Cells

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

scholarly journals Solubility Parameters of Amino Acids on Liquid–Liquid Phase Separation and Aggregation of Proteins

2021 ◽  
Vol 9 ◽  
Author(s):  
Akira Nomoto ◽  
Suguru Nishinami ◽  
Kentaro Shiraki
Keyword(s):  
Amino Acids ◽  
Phase Separation ◽  
Amino Acid ◽  
Liquid Phase ◽  
Aromatic Amino Acids ◽  
Liquid Phase Separation ◽  
Solubility Parameters ◽  
Liquid Liquid Phase Separation

The solution properties of amino acids determine the folding, aggregation, and liquid–liquid phase separation (LLPS) behaviors of proteins. Various indices of amino acids, such as solubility, hydropathy, and conformational parameter, describe the behaviors of protein folding and solubility both in vitro and in vivo. However, understanding the propensity of LLPS and aggregation is difficult due to the multiple interactions among different amino acids. Here, the solubilities of aromatic amino acids (SAs) were investigated in solution containing 20 types of amino acids as amino acid solvents. The parameters of SAs in amino acid solvents (PSASs) were varied and dependent on the type of the solvent. Specifically, Tyr and Trp had the highest positive values while Glu and Asp had the lowest. The PSAS values represent soluble and insoluble interactions, which collectively are the driving force underlying the formation of droplets and aggregates. Interestingly, the PSAS of a soluble solvent reflected the affinity between amino acids and aromatic rings, while that of an insoluble solvent reflected the affinity between amino acids and water. These findings suggest that the PSAS can distinguish amino acids that contribute to droplet and aggregate formation, and provide a deeper understanding of LLPS and aggregation of proteins.

scholarly journals Liquid-liquid phase separation of tau driven by hydrophobic interaction facilitates fibrillization of tau

2020 ◽  
Author(s):  
Yanxian Lin ◽  
Yann Fichou ◽  
Andrew P. Longhini ◽  
Luana C. Llanes ◽  
Yinson Yin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Tau Protein ◽  
Liquid Phase Separation ◽  
Complex Coacervation ◽  
Amyloid Aggregation ◽  
Apparent Conflict ◽  
Liquid Liquid Phase Separation

AbstractAmyloid aggregation of tau protein is implicated in neurodegenerative diseases, yet its facilitating factors are poorly understood. Recently, tau has been shown to undergo liquid liquid phase separation (LLPS) both in vivo and in vitro. LLPS was shown to facilitate tau amyloid aggregation in certain cases, while independent of aggregation in other cases. It is therefore important to understand the differentiating properties that resolve this apparent conflict. We report on a model system of hydrophobically driven LLPS induced by high salt concentration (LLPS-HS), and compare it to electrostatically driven LLPS represented by tau-RNA/heparin complex coacervation (LLPS-ED). We show that LLPS-HS promotes tau protein dehydration, undergoes maturation and directly leads to canonical tau fibrils, while LLPS-ED is reversible, remains hydrated and does not promote amyloid aggregation. We show that the nature of the interaction driving tau condensation is the differentiating factor between aggregation-prone and aggregation-independent LLPS.

scholarly journals Abstract P-24: Microscopic Analyses of Liquid-Liquid Phase Separation Induced by Linker Histone H1.0

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S21-S22
Author(s):  
Olga Geraskina ◽  
Natalya Maluchenko ◽  
Vasily Studitsky ◽  
Nadezhda Gerasimova ◽  
Daria Koshkina ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Dynamic Properties ◽  
Chromatin Condensation ◽  
Liquid Phase Separation ◽  
Moderate Rate ◽  
Condensate Formation ◽  
Dividing Cells ◽  
Liquid Liquid Phase Separation

Background: Liquid-liquid phase separation (LLPS) that leads to the formation of temporary functional domains in cells plays an important role in the processes of chromatin condensation and gene regulation. Earlier, it was demonstrated that histone H1.4 can form LLPS droplets with DNA. In the present work, LLPS was studied for histone H1.0, which is mainly expressed in differentiated and non-dividing cells. H1.0 is involved in cancer development: its amount decreases with the progression of tumor cells to malignancy. Methods: LSM710 confocal microscope (Zeiss) equipped with the 40x/1.2W objective was used to image mixtures of H1.0 with Cy3/Cy5 labeled DNA or nucleosomes in fluorescent and transmitted-light channels at the excitation of 514 nm. The formation of condensates as a result of LLPS was confirmed by salt-jump and FRAP/FLIP experiments. Results: Condensates were not observed when the ratio of negative to positive charges (N/P) in the samples was >1. At N/P~0.7, optically homogeneous droplet-like condensates were found. The appearance of condensates, their size and shape depended on concentrations of H1.0 and DNA. LLPS condensates but not aggregates disappeared by salt-jump to 650 mM NaCl. FRAP/FLIP experiments revealed a moderate rate of fluorescence recovery (τ½22s) indicating moderate DNA mobility of the H1.0-mediated condensates. The appearance of condensates was also observed in the mixtures of H1.0, DNA and Cy3/Cy5-labeled nucleosomes. Nucleosomes were involved in the condensate formation and found to be 2-fold more mobile (τ½10 s) than DNA. Conclusion: LLPS-related properties of H1.0 were studied for DNA and nucleosomes in vitro. Comparison with H1.4 shows that H1.0 forms liquid condensates of approximately the same size. Our result also may indicate that chromatin retains pronounced dynamic properties in H1.0-induced droplets despite the fact that H1.0 induces the formation of more compact chromatin.

scholarly journals Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation

2020 ◽  
Vol 117 (31) ◽  
pp. 18540-18549 ◽  
Author(s):  
Anne-Marie Ladouceur ◽  
Baljyot Singh Parmar ◽  
Stefan Biedzinski ◽  
James Wall ◽  
S. Graydon Tope ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Bulk Diffusion ◽  
Liquid Phase Separation ◽  
Bacterial Cells ◽  
Liquid Liquid Phase Separation

Once described as mere “bags of enzymes,” bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid–liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) inEscherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

scholarly journals Liquid–Liquid Phase Separation in the Presence of Macromolecular Crowding and State-Dependent Kinetics

2021 ◽  
Vol 22 (13) ◽  
pp. 6675
Author(s):  
Alick-O. Vweza ◽  
Chul-Gyu Song ◽  
Kil-To Chong
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Late Stage ◽  
Macromolecular Crowding ◽  
Liquid Phase Separation ◽  
Self Organization ◽  
State Dependent ◽  
Liquid Liquid Phase Separation

Biomolecular condensates formed via liquid–liquid phase separation (LLPS) are increasingly being shown to play major roles in cellular self-organization dynamics in health and disease. It is well established that macromolecular crowding has a profound impact on protein interactions, particularly those that lead to LLPS. Although synthetic crowding agents are used during in vitro LLPS experiments, they are considerably different from the highly crowded nucleo-/cytoplasm and the effects of in vivo crowding remain poorly understood. In this work, we applied computational modeling to investigate the effects of macromolecular crowding on LLPS. To include biologically relevant LLPS dynamics, we extended the conventional Cahn–Hilliard model for phase separation by coupling it to experimentally derived macromolecular crowding dynamics and state-dependent reaction kinetics. Through extensive field-theoretic computer simulations, we show that the inclusion of macromolecular crowding results in late-stage coarsening and the stabilization of relatively smaller condensates. At a high crowding concentration, there is an accelerated growth and late-stage arrest of droplet formation, effectively resulting in anomalous labyrinthine morphologies akin to protein gelation observed in experiments. These results not only elucidate the crowder effects observed in experiments, but also highlight the importance of including state-dependent kinetics in LLPS models, and may help in designing further experiments to probe the intricate roles played by LLPS in self-organization dynamics of cells.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Taurine suppresses liquid-liquid phase separation of lysozyme protein

2021 ◽  
Author(s):  
Kanae Tsubotani ◽  
Sayuri Maeyama ◽  
Shigeru Murakami ◽  
Stephen W Schaffer ◽  
Takashi Ito
Keyword(s):  
Phase Separation ◽  
Polyethylene Glycol ◽  
Critical Temperature ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Compatible Osmolyte ◽  
Hen Egg ◽  
Liquid Liquid Phase Separation ◽  
In Cells

AbstractTaurine is a compatible osmolyte that infers stability to proteins. Recent studies have revealed that liquid-liquid phase separation (LLPS) of proteins underlie the formation of membraneless organelles in cells. In the present study, we evaluated the role of taurine on LLPS of hen egg lysozyme. We demonstrated that taurine decreases the turbidity of the polyethylene glycol-induced crowding solution of lysozyme. We also demonstrated that taurine attenuates LLPS-dependent cloudiness of lysozyme solution with 0.5 or 1M NaCl at a critical temperature. Moreover, we observed that taurine inhibits LLPS formation of a heteroprotein mix solution of lysozyme and ovalbumin. These data indicate that taurine can modulate the formation of LLPS of proteins.

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