Undergo Phase Separation Research Articles

Peptides for Liquid-Liquid Phase Separation: An Emerging Biomaterial.

Liquid-liquid phase separation (LLPS) refers to a spontaneous separation behavior of biomacromolecules under specific physiological conditions, playing a crucial role in regulating various biological processes. Recent advances in synthetic peptides have greatly improved our understanding of peptide-based coacervate droplets and expanded their applications in biomedicine. Numerous peptide sequences have been reported that undergo phase separation, enabling the concentration and sequestration of different guest molecules for purposes such as drug delivery, catalytic performance, and bioanalytical techniques. Particularly, some of these peptides offer significant advantages in controlled drug release, efficient cell transfection, accelerated reaction kinetics, and selective biomarker detection. This review provides an overview of recent developments in peptide-based LLPS, exploring various strategies for designing peptide sequences and their biomedical applications. It also addresses the challenges and future directions for LLPS peptide vehicles as promising biomaterials.

A scale-invariant log-normal droplet size distribution below the critical concentration for protein phase separation

Many proteins have been recently shown to undergo a process of phase separation that leads to the formation of biomolecular condensates. Intriguingly, it has been observed that some of these proteins form dense droplets of sizeable dimensions already below the critical concentration, which is the concentration at which phase separation occurs. To understand this phenomenon, which is not readily compatible with classical nucleation theory, we investigated the properties of the droplet size distributions as a function of protein concentration. We found that these distributions can be described by a scale-invariant log-normal function with an average that increases progressively as the concentration approaches the critical concentration from below. The results of this scaling analysis suggest the existence of a universal behaviour independent of the sequences and structures of the proteins undergoing phase separation. While we refrain from proposing a theoretical model here, we suggest that any model of protein phase separation should predict the scaling exponents that we reported here from the fitting of experimental measurements of droplet size distributions. Furthermore, based on these observations, we show that it is possible to use the scale invariance to estimate the critical concentration for protein phase separation.

Surface-induced nano-generator utilizing a thermo-responsive smart window based on ionic liquid aqueous solution that exhibits lower critical solution temperature type phase separation

We demonstrate a surface-induced nano-generator utilizing a thermo-responsive smart window based on ionic liquid aqueous solution that exhibits lower critical solution temperature (LCST) type phase separation. This smart window was fabricated by filling an aqueous solution of [nBu4P][CF3COO] between the glass substrates coated with two different polymers: a polyimide with an alkyl side chain and an amorphous fluoropolymer. Below the LCST, the transmittance of the smart window was 87 %, nearly identical to that of a glass substrate. In contrast, when heated above the LCST, the [nBu4P][CF3COO] aqueous solution undergoes phase separation, causing the [nBu4P] cations and [CF3COO] anions to adsorb onto the polyimide with the alkyl side chain and the amorphous fluoropolymer facilitated by the surface pinning effect. This adsorption process results in the smart window generating electricity while transitioning to an opaque state. Therefore, the proposed smart window functions as an electricity-generating thermo-responsive device that can switch between transparent and opaque states in response to temperature changes.

The landscape of intrinsically disordered proteins in Leishmania parasite: Implications for drug discovery

Proteins that lack three-dimensional structures are known as Intrinsically disordered proteins (IDPs). In this study, we aimed to identify intrinsically disordered proteins in the Leishmania donovani proteome using various predictors that can identify IDPs based on amino acid residues and charge hydropathy. Top identified IDPs are analyzed using STRING, PSP-Hunter, Deep Loc-2.0, and Alpha fold to understand the protein-protein interaction, phase separation, localization, and structural assessment of those proteins. From this study, we found that >50 % of Leishmania donovani proteome has proteins or regions of proteins that are intrinsically disordered with VSL2 score >0.5; most proteins interact with many other proteins with PPI enrichment p-value <1.0e-16. Few proteins, such as Protein phosphatase inhibitor, UMSBP, and Zinc knuckle, have redox-sensitive regions. Functional disorder profiles of identified IDPs showed MoRFs and predicted protein domains. HASPB, UTP11, Nucleolar protein 12, and UMSBP have a high probability of undergoing phase separation. Localization studies showed that most of these proteins are in the cytoplasm and nucleus. Our present study of identifying IDPs in Leishmania proteome yields significant information on druggable targets and can serve as a basis for further studies to identify unexplored pathways.

Predicting the sequence-dependent backbone dynamics of intrinsically disordered proteins.

How the sequences of intrinsically disordered proteins (IDPs) code for functions is still an enigma. Dynamics, in particular residue-specific dynamics, holds crucial clues. Enormous efforts have been spent to characterize residue-specific dynamics of IDPs, mainly through NMR spin relaxation experiments. Here, we present a sequence-based method, SeqDYN, for predicting residue-specific backbone dynamics of IDPs. SeqDYN employs a mathematical model with 21 parameters: one is a correlation length and 20 are the contributions of the amino acids to slow dynamics. Training on a set of 45 IDPs reveals aromatic, Arg, and long-branched aliphatic amino acids as the most active in slow dynamics whereas Gly and short polar amino acids as the least active. SeqDYN predictions not only provide an accurate and insightful characterization of sequence-dependent IDP dynamics but may also serve as indicators in a host of biophysical processes, including the propensities of IDP sequences to undergo phase separation.

Predicting the sequence-dependent backbone dynamics of intrinsically disordered proteins

How the sequences of intrinsically disordered proteins (IDPs) code for functions is still an enigma. Dynamics, in particular residue-specific dynamics, holds crucial clues. Enormous efforts have been spent to characterize residue-specific dynamics of IDPs, mainly through NMR spin relaxation experiments. Here, we present a sequence-based method, SeqDYN, for predicting residue-specific backbone dynamics of IDPs. SeqDYN employs a mathematical model with 21 parameters: one is a correlation length and 20 are the contributions of the amino acids to slow dynamics. Training on a set of 45 IDPs reveals aromatic, Arg, and long-branched aliphatic amino acids as the most active in slow dynamics whereas Gly and short polar amino acids as the least active. SeqDYN predictions not only provide an accurate and insightful characterization of sequence-dependent IDP dynamics but may also serve as indicators in a host of biophysical processes, including the propensities of IDP sequences to undergo phase separation.

Studies of Protein Phase Separation Using Leishmania Kinetoplastid Membrane Protein-11.

Despite the significant understanding of phase separation in proteins with intrinsically disordered regions, a considerable percentage of proteins without such regions also undergo phase separation, presenting an intriguing area for ongoing research across all kingdoms of life. Using a combination of spectroscopic and microscopic techniques, we report here for the first time that a low temperature and low pH can trigger the liquid-liquid phase separation (LLPS) of a parasitic protein, kinetoplastid membrane protein-11 (KMP-11). Electrostatic and hydrophobic forces are found to be essential for the formation and stability of phase-separated protein assemblies. We show further that the increase in the ionic strength beyond a threshold decreases the interchain electrostatic interactions acting between the alternate charged blocks, altering the propensity for phase separation. More interestingly, the addition of cholesterol inhibits LLPS by engaging the cholesterol recognition amino acid consensus (CRAC)-like domains present in the protein. This was further confirmed using a CRAC-deleted mutant with perturbed cholesterol binding, which did not undergo LLPS.

YTHDF2 phase separation promotes arsenic-induced keratinocyte transformation in a poly-m6A-dependent manner by inhibiting translational initiation of the key tumor suppressor PTEN

The phase separation of N6-methyladenosine (m6A) binding protein YTHDF2 plays a vital role in arsenic-induced skin damage, and YTHDF2 can bind to m6A-methylated mRNA of tumor suppressor PTEN. However, whether and how YTHDF2 phase separation regulates PTEN involved in arsenic-induced malignant transformation of keratinocytes remains blank. Here, we established arsenite-induced transformation models with stable expression of wild-type YTHDF2 or mutant YTHDF2 protein in vitro and in vivo. We found that the YTHDF2 protein underwent phase separation during arsenite-induced malignant transformation of keratinocytes, and YTHDF2 phase separation promoted the malignant phenotype of keratinocytes. Mechanically, YTHDF2 phase separation reduced PTEN protein levels, which in turn activated the pro-survival AKT signal. The binding of YTHDF2 to multiple m6A sites on PTEN mRNA drove YTHDF2 phase separation, inhibiting PTEN translation initiation and thus reducing PTEN protein levels. YTHDF2 phase separation recruited translation-initiation-factor kinase EIF2AK1 to phosphorylate eIF2α, thereby inhibiting translation initiation of poly-m6A-methylated PTEN mRNA. Furthermore, arsenite-induced oxidative stress triggered YTHDF2 phase separation by increasing m6A levels of PTEN mRNA. Our results demonstrated that YTHDF2 phase separation promotes arsenite-induced malignant transformation by inhibiting PTEN translation in a poly-m6A-dependent manner. This study sheds light on arsenic carcinogenicity from the novel aspect of m6A-mediated YTHDF2 phase separation.

A coarse-grained model for disordered and multi-domain proteins.

Many proteins contain more than one folded domain, and such modular multi-domain proteins help expand the functional repertoire of proteins. Because of their larger size and often substantial dynamics, it may be difficult to characterize the conformational ensembles of multi-domain proteins by simulations. Here, we present a coarse-grained model for multi-domain proteins that is both fast and provides an accurate description of the global conformational properties in solution. We show that the accuracy of a one-bead-per-residue coarse-grained model depends on how the interaction sites in the folded domains are represented. Specifically, we find excessive domain-domain interactions if the interaction sites are located at the position of the Cα atoms. We also show that if the interaction sites are located at the center of mass of the residue, we obtain good agreement between simulations and experiments across a wide range of proteins. We then optimize our previously described CALVADOS model using this center-of-mass representation, and validate the resulting model using independent data. Finally, we use our revised model to simulate phase separation of both disordered and multi-domain proteins, and to examine how the stability of folded domains may differ between the dilute and dense phases. Our results provide a starting point for understanding interactions between folded and disordered regions in proteins, and how these regions affect the propensity of proteins to self-associate and undergo phase separation.

Clustering of RNA Polymerase II C-Terminal Domain Models upon Phosphorylation.

RNA polymerase II (Pol II) C-terminal domain (CTD) is known to have crucial roles in regulating transcription. CTD has also been highly recognized for undergoing phase separation, which is further associated with its regulatory functions. However, the molecular interactions that the CTD forms to induce clustering to drive phase separations and how the phosphorylation of the CTD affects clustering are not entirely known. In this work, we studied the concentrated solutions of two heptapeptide repeat (2CTD) models at different phosphorylation patterns and protein and ion concentrations using all-atom molecular dynamics simulations to investigate clustering behavior and molecular interactions driving the cluster formation. Our results show that salt concentration and phosphorylation patterns play an important role in determining the clustering pattern, specifically at low protein concentrations. The balance between inter- and intrapeptide interactions and counterion coordination together impact the clustering behavior upon phosphorylation.

Dynamic Light Scattering Microrheology of Phase-Separated Poly(vinyl) Alcohol–Phytagel Blends

In this investigation, we explored the microrheological characteristics of dilute hydrogels composed exclusively of Poly(vinyl) alcohol (PVA), Phytagel (PHY), and a blend of the two in varying concentrations. Each of these polymers has established applications in the biomedical field, such as drug delivery and lens drops. This study involved varying the sample concentrations from 0.15% to 0.3% (w/w) to assess how the concentration influenced the observed rheological response. Two probe sizes were employed to examine the impact of the size and verify the continuity hypothesis. The use of two polymer blends revealed their immiscibility and tendency to undergo phase separation, as supported by the existing literature. Exploring the microrheological structure is essential for a comprehensive understanding of the molecular scale. Dynamic light scattering (DLS) was chosen due to its wide frequency range and widespread availability. The selected dilute concentration range was hypothesized to fall within the transition from an ergodic to a non-ergodic medium. Properly identifying the sample’s nature during an analysis—whether it is ergodic or not—is critical, as highlighted in the literature. The obtained results clearly demonstrate an overlap in the results for the storage (G’) and loss moduli (G″) for the different probe particle sizes, confirming the fulfillment of the continuum hypothesis.

Evidence for biomolecular condensates of MatP in spatiotemporal regulation of the bacterial cell division cycle.

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP ( matS ), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatP's role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

Simulation of lithium hydroxide decomposition using deep potential molecular dynamics.

Chemical reactions and vapor-liquid equilibria for molten lithium hydroxide (LiOH) were studied using molecular dynamics simulations and a deep potential (DP) model. The neural network for the model was trained on quantum density functional theory data for a range of conditions. The DP model allows simulations over timescales of hundreds of ns, which provide equilibrium compositions for the systems of interest. Single-phase NPT simulations of the liquid show the decomposition of LiOH into lithium oxide (Li2O) and dissolved water (H2O). These DP results were validated by direct abinitio molecular dynamics simulations that confirmed the accuracy of the model with respect to reaction kinetics and equilibrium properties of the melt. The reactive vapor-liquid behavior of this system was subsequently studied using direct coexistence interfacial DP simulations. Partial pressures of H2O in the vapor are found to be in close agreement with available experimental measurements. By fitting temperature-dependent expressions for the reaction equilibrium and Henry's law constants, the equilibrium composition for any given initial composition and temperature can be quantitatively modeled. For high initial concentrations of Li2O or H2O, mixtures of LiOH + Li2O/H2O are found to undergo phase separation. The present study illustrates how DP-based molecular dynamics simulations can be used for quantitative modeling of multiphase reactive behavior with the accuracy of the underlying abinitio quantum chemical methods.

Plant PR1 rescues condensation of the plastid iron-sulfur protein by a fungal effector.

Plant pathogens secrete numerous effectors to promote host infection, but whether any of these toxic proteins undergoes phase separation to manipulate plant defence and how the host copes with this event remain elusive. Here we show that the effector FolSvp2, which is secreted from the fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), translocates a tomato iron-sulfur protein (SlISP) from plastids into effector condensates in planta via phase separation. Relocation of SlISP attenuates plant reactive oxygen species production and thus facilitates Fol invasion. The action of FolSvp2 also requires K205 acetylation that prevents ubiquitination-dependent degradation of this protein in both Fol and plant cells. However, tomato has evolved a defence protein, SlPR1. Apoplastic SlPR1 physically interacts with and inhibits FolSvp2 entry into host cells and, consequently, abolishes its deleterious effect. These findings reveal a previously unknown function of PR1 in countering a new mode of effector action.

Molybdenite Re-Os geochronology and conditions of formation of potassic and sodic-calcic alteration associated with the Plaka porphyry Mo-Cu system, Lavrion, Greece

The Plaka porphyry Mo-Cu system occurs in the world-class Lavrion Ag-Pb-Zn district in Attica, southern Greece. It is spatially associated with a granodiorite porphyry that intruded the Attic-Cycladic Crystalline Complex in the late Miocene, along the footwall of the Western Cycladic detachment fault. A Re-Os age of 9.51 ± 0.04 Ma indicates that molybdenite formed during the early stage of the granodiorite porphyry intrusion and that subsequent cooling was very rapid. Brittle deformation and hydrothermal fluid flow created a network of A-, B-, diopside-actinolite and D- veins, associated with potassic-, sodic-calcic- and sericitic alterations. Potassic alteration is characterized by secondary biotite + K-feldspar + quartz + magnetite ± apatite, contains disseminated molybdenite, pyrite, and chalcopyrite, and formed at 420–500 °C, at pressures up to 530 bars (< 5.3 km depth) from hydrothermal fluids that underwent phase separation. Sodic-calcic alteration is devoid of Cu-Mo mineralization and, consists of diopside + actinolite + oligoclase/andesine + titanite + magnetite ± epidote-allanite ± chlorite ± quartz, which corresponded to a temperature range of between 350 and < 500 °C. Primary magnetite, titanite and biotite crystallized between the nickel‑nickel oxide (NNO) and hematite-magnetite (HM) buffers, indicating fairly oxidizing conditions for the granodioritic magma. Hydrothermal biotite plots closer to the HM buffer suggesting increasing oxygen fugacity during exsolution of the hydrothermal fluids associated with potassic alteration. The system evolved toward more reducing conditions during sericitic alteration and associated pyrite-molybdenite mineralization. A combination of evaporated seawater and magmatic fluids likely caused formation of the sodic-calcic alteration through the decarbonation of the host marble.

The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis and maintain translational repression of maternal mRNA and oocyte quality.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo Caenorhabditis elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the chaperonin-containing tailless complex polypeptide 1 (CCT) chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of endoplasmic reticulum sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer.

Carboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Noise-Induced Phase Separation and Time Reversal Symmetry Breaking in Active Field Theories Driven by Persistent Noise.

Within the Landau-Ginzburg picture of phase transitions, scalar field theories develop phase separation because of a spontaneous symmetry-breaking mechanism. This picture works in thermodynamics but also in the dynamics of phase separation. Here we show that scalar nonequilibrium field theories undergo phase separation just because of nonequilibrium fluctuations driven by a persistent noise. The mechanism is similar to what happens in motility-induced phase separation where persistent motion introduces an effective attractive force. We observe that noise-induced phase separation occurs in a region of the phase diagram where disordered field configurations would otherwise be stable at equilibrium. Measuring the local entropy production rate to quantify the time-reversal symmetry breaking, we find that such breaking is concentrated on the boundary between the two phases.

A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation.

Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane-bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins' phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin-3, a widely studied lysosomal damage marker, is driven by cation-π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π-π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion-like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation-π, π-π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion-like IDRs.