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Liquid–liquid phase separation (LLPS) is associated with the spatiotemporal organization of the autophagic machinery, cell migration signaling, and stress granule dynamics. The proven functional spectrum of Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) in LLPS events includes autophagy, stress granule disassembly, and focal adhesion assembly. Phase separation underlies aspects of other cellular process that are regulated by ULK/Atg1 (e.g., cell proliferation and cell death pathways, endoplasmic reticulum-to-Golgi trafficking), but whether ULK/Atg1 activity influences LLPS in these contexts remains to be determined. Autophagy – a highly regulated intracellular degradation process – is pivotal in maintaining cellular homeostasis. Liquid–liquid phase separation (LLPS) is a fundamental mechanism regulating the formation and function of membrane-less compartments. Recent research has unveiled connections between LLPS and autophagy, suggesting that phase separation events may orchestrate the spatiotemporal organization of autophagic machinery and cargo sequestration. The Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) family of proteins is best known for its regulatory role in initiating autophagy, but there is growing evidence that the functional spectrum of ULK/Atg1 extends beyond autophagy regulation. In this review, we explore the spatial and temporal regulation of the ULK/Atg1 family of kinases, focusing on their recruitment to LLPS-driven compartments, and highlighting their multifaceted functions beyond their traditional role. [ABSTRACT FROM AUTHOR]

Liquid-liquid phase separation (LLPS) regulates many biological processes including RNA metabolism, chromatin rearrangement, and signal transduction. Aberrant LLPS potentially leads to serious diseases. Therefore, the identification of the LLPS proteins is crucial. Traditionally, biochemistry-based methods for identifying LLPS proteins are costly, time-consuming, and laborious. In contrast, artificial intelligence-based approaches are fast and cost-effective and can be a better alternative to biochemistry-based methods. Previous research methods employed word2vec in conjunction with machine learning or deep learning algorithms. Although word2vec captures word semantics and relationships, it might not be effective in capturing features relevant to protein classification, like physicochemical properties, evolutionary relationships, or structural features. Additionally, other studies often focused on a limited set of features for model training, including planar π contact frequency, pi-pi, and β-pairing propensities. To overcome such shortcomings, this study first constructed a reliable dataset containing 1206 protein sequences, including 603 LLPS and 603 non-LLPS protein sequences. Then a computational model was proposed to efficiently identify the LLPS proteins by perceiving semantic information of protein sequences directly; using an ESM2-36 pre-trained model based on transformer architecture in conjunction with a convolutional neural network. The model could achieve an accuracy of 85.68% and 89.67%, respectively on training data and test data, surpassing the accuracy of previous studies. The performance demonstrates the potential of our computational methods as efficient alternatives for identifying LLPS proteins. [ABSTRACT FROM AUTHOR]

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described. [ABSTRACT FROM AUTHOR]

Liquid ‒ liquid phase separation (LLPS) is a general phenomenon recently recognized to be critically involved in the regulation of a variety of cellular biological processes, such as transcriptional regulation, heterochromatin formation and signal transduction, through the compartmentalization of proteins or nucleic acids into droplet-like condensates. These processes are directly or indirectly related to tumor initiation and treatment. Posttranslational modifications (PTMs), which represent a rapid and reversible mechanism involved in the functional regulation of proteins, have emerged as key events in modulating LLPS under physiological or pathophysiological conditions, including tumorigenesis and antitumor therapy. In this review, we introduce the biological functions participated in cancer-associated LLPS, discuss the potential roles of LLPS during tumor onset or therapy, and emphasize the mechanistic characteristics of LLPS regulated by PTMs and its effects on tumor progression. We then provide a perspective on further studies on LLPS and its regulation by PTMs in cancer research. This review aims to broaden the understanding of the functions of LLPS and its regulation by PTMs under normal or aberrant cellular conditions. • Tumor-related LLPS can be finely tuned by PTMs. • The progression of tumors appears to be intricately linked with aberrant LLPS. • PTMs regulated-LLPS play an important role in cancer initiation and clinical tumor treatment. [ABSTRACT FROM AUTHOR]

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (k D) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Polyamide 11 microparticles produced via phase separation & precipitation. • Cloud point and solution temperature curve of the PA11-ethanol system. • Control of particle size by choice of process conditions. • Characterization of product particles (structure, thermal & bulk solid properties). • SLS processability and layer adhesion is demonstrated by multi-layer specimen. Abstract Within this contribution liquid-liquid phase separation (LLPS) and precipitation from ethanol was studied as an approach to produce polyamide 11 (PA11) powders for selective laser sintering (SLS). To this end, the cloud point and solution temperature curve of the PA11-ethanol system was determined experimentally via turbidity measurements. The proper range of system composition and temperature for particle formation was deduced. The dependence of particle characteristics on process parameters (polymer concentration, stirring conditions and temperature regime) during LLPS and precipitation was assessed and the products were characterized with respect to their size and morphology. Furthermore, structural, rheological and thermal characteristics were analyzed and correlated with process parameters. Molecular weight distributions were determined. After removal of fines and dry coating with hydrophobic fumed silica, an optimized PA11 powder with mean particle size of 91 µm showing good flowability for SLS was obtained. SLS processability of this optimized PA11 powder was demonstrated by building multi-layered test specimens in a laser sintering machine. With this contribution, we present a comprehensive workflow for the process development, product characterization and product application of a SLS powder manufactured via precipitation. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs), which are among the most well-characterized noncoding RNAs, have attracted much attention due to their regulatory functions and potential therapeutic options in many types of disease. Liquid–liquid phase separation (LLPS), the formation of droplet condensates, is involved in various cellular processes, but the molecular interactions of lncRNAs in LLPS are unclear. In this review, we describe the research development on LLPS, including descriptions of various methods established to identify LLPS, summarize the physiological and pathological functions of LLPS, identify the molecular interactions of lncRNAs in LLPS, and present the potential applications of leveraging LLPS in the clinic. The aim of this review is to update the knowledge on the association between LLPS and lncRNAs, which might provide a new direction for the treatment of LLPS-mediated disease. • Liquid–liquid phase separation has diverse biological functions. • This review synthesizes how the lncRNAs may impact liquid–liquid phase separation. • Providing insights into potential applications leveraging liquid–liquid phase separation in the clinic. [ABSTRACT FROM AUTHOR]

Membrane vesiculation is an energy-costing process. Previous studies paid much attention to proteins with curvature-inducing motifs. Recent publications reveal that the liquid-like protein assembly on membrane surfaces provides an efficient yet structure-independent mechanism for increasing the membrane curvature, which plays important roles in vesicle transport in many aspects. Intrinsically disordered regions (IDRs) within the proteins are highly potent drivers of membrane curvature by providing large hydrodynamic radii to generate steric pressure. Biomolecular condensates formed by phase separation can provide a reaction platform for sequential processes or generate a wetting surface to sequestrate cargos and trigger membrane remodeling. We review the latest progress in yeast and mammalian cells, focus on the mechanism of clathrin-mediated endocytosis (CME) and autophagy initiation, and compare with what we know in model plant Arabidopsis. The comparison may give important insights into the understanding of basic membrane trafficking mechanisms in plant cells. [ABSTRACT FROM AUTHOR]

"Membraneless organelles," also referred to as biomolecular condensates, perform a variety of cellular functions and their dysregulation is implicated in cancer and neurodegeneration. In the last two decades, liquid-liquid phase separation (LLPS) of intrinsically disordered and multidomain proteins has emerged as a plausible mechanism underlying the formation of various biomolecular condensates. Further, the occurrence of liquid-to-solid transitions within liquid-like condensates may give rise to amyloid structures, implying a biophysical link between phase separation and protein aggregation. Despite significant advances, uncovering the microscopic details of liquid-to-solid phase transitions using experiments remains a considerable challenge and presents an exciting opportunity for the development of computational models which provide valuable, complementary insights into the underlying phenomenon. In this review, we first highlight recent biophysical studies which provide new insights into the molecular mechanisms underlying liquid-to-solid (fibril) phase transitions of folded, disordered and multi-domain proteins. Next, we summarize the range of computational models used to study protein aggregation and phase separation. Finally, we discuss recent computational approaches which attempt to capture the underlying physics of liquid-to-solid transitions along with their merits and shortcomings. [Display omitted] [ABSTRACT FROM AUTHOR]

The control of supramolecular complexes in living systems at the molecular level is an important goal in life-sciences. Spatiotemporal organization of molecular distribution & flow of such complexes are essential physicochemical processes in living cells and important for pharmaceutical processes. Membraneless organelles (MO) found in eukaryotic cells, formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins (IDPs) control and adjust intracellular organization. Artificially designed compartments based on LLPS open up a novel pathway to control chemical flux and partition in vitro and in vivo. We designed a library of chemically precisely defined block copolymer-like proteins based on elastin-like proteins (ELPs) with defined charge distribution and type, as well as polar and hydrophobic block domains. This enables the programmability of physicochemical properties and to control adjustable LLPS in vivo attaining control over intracellular partitioning and flux as role model for in vitro and in vivo applications. Tailor-made ELP-like block copolymer proteins exhibiting IDP-behavior enable LLPS formation in vitro and in vivo allowing the assembly of membrane-based and membraneless superstructures via protein phase-separation in E. coli. Subsequently, we demonstrate the responsiveness of protein phase-separated spaces (PPSSs) to environmental physicochemical triggers and their selective, charge-dependent and switchable interaction with DNA or extrinsic and intrinsic molecules enabling their selective shuttling across semipermeable phase boundaries including (cell)membranes. This paves the road for adjustable artificial PPSS-based storage and reaction spaces and the specific transport across phase boundaries for applications in pharmacy and synthetic biology. • An amphiphilic protein (aELP) library allows for adjustable compartments in vivo. • Extrinsic stimuli modulate protein-phase separated spaces (PPSS) in E. coli. • PPSS formed by charged aELPs direct DNA localization. • Charged aELP forming PPSS can control molecular flux of molecules in vivo. • Responsive PPSS can be used in pharmaceutical applications and synthetic biology. [ABSTRACT FROM AUTHOR]

Turbid elution pools and high column back pressure are common during elution of monoclonal antibodies (mAbs) by acidic pH in Protein A chromatography. This phenomenon has been historically attributed to acid-induced precipitation of incorrectly folded or pH-sensitive mAbs and host cell proteins (HCPs). In this work, we propose a new mechanism that may account for some observations of elution turbidity in Protein A chromatography. We report several examples of turbidity and high column back pressure occurring transiently under a short course of neutral conditions during Protein A elution. A systematic study of three mAbs displaying this behavior revealed phase separation characterized by liquid drops under certain conditions including neutral pH, low ionic strength, and high protein concentration. These liquid droplets caused solution turbidity and exhibited extremely high viscosity, resulting in high column back pressure. We found out that the droplets were formed through liquid–liquid phase separation (LLPS) as a result of protein self-association. We also found multiple factors, including pH, temperature, ionic strength, and protein concentration can affect LLPS behaviors. Careful selection of process parameters during protein A elution, including temperature, flow rate, buffer, and salt can inhibit formation of a dense liquid phase, reducing both turbidity (by 90%) and column back pressure (below 20 pounds per square inch). These findings provide both mechanistic insight and practical mitigation strategies for Protein A chromatography induced LLPS. [ABSTRACT FROM AUTHOR]

[Display omitted] We describe a lattice model to analyze the effect of protein surface charge distributions on protein solubility, aggregation, and condensation. Surface charges were modeled by considering a protein as a cubic particle with each face covered by two negative and positive patches (net zero charge). Particles were moved to adjacent lattice points according to a Monte Carlo algorithm. We observed that proteins having the same isoelectric point can have distinct aggregation propensities, depending on surface charge arrangements. Furthermore, with regard to the effect of heterogeneous intracellular environment, mixing two particle types affected the aggregation properties of the individual type. [ABSTRACT FROM AUTHOR]

By lyophilization of rifampicin (RIF) solution in TBA/water with various solvent compositions, uniform and amorphous rifampicin (RIF) microspheres were produced. Using 55% TBA solution, the obtained RIF microspheres have a mono-dispersive size distribution with diameters range from 1 to 3 μm. The RIF microspheres are found to be amorphous by X-ray diffraction, and are expected to dissolve much faster than the crystalline RIF upon inhalation. Mechanistic investigation revealed that the amorphous RIF microspheres were formed due to liquid–liquid phase separation (LLPS) occurred during the freezing of the TBA/water solution. We also observed that the RIF microspheres can be readily phagocytized by activated THP-1 cells within 15 min. The suitable size distribution, high solubility, and readiness for phagocytosis by macrophages, all suggest that the lyophilized amorphous RIF microspheres could be potentially used as an anti-tuberculosis inhalation therapy. In addition, similar process was used to lyophilize TBA/water solutions of several other drugs, including rifaximin, rifapentine, paclitaxel, and isoniazid. We found that for drugs with appropriate physiochemical properties, such as paclitaxel and rifaximin, mono-dispersive microspheres could be obtained as well, which demonstrated that freezing induced LLPS could be utilized as a novel particle engineering methodology to produce drug microspheres by lyophilization. [ABSTRACT FROM AUTHOR]

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α -Synuclein (α Syn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of α Syn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13C-labeled His-50 of α Syn, we show that the distribution of the α Syn conformation is skewed from the initial completely random state to a loose β -rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose β -rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets. • α -Synuclein forms phase-separated liquid droplets in molecular crowding condition. • Evolution of conformational ensemble within early-stage droplet was studied. • Auto-inhibitory form of α Syn is released upon droplet formation. • α Syn forms a loose β -rich assembly in the droplet that grows into amyloid fibrils. • Dynamic nuclear polarization-enhanced solid-state NMR is a key tool for such study. [ABSTRACT FROM AUTHOR]

Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates. [ABSTRACT FROM AUTHOR]

The development of a novel polybutylene terephthalate (PBT) feedstock material for powder bed fusion of polymers with laser beam (PBF-LB/P) by means of liquid-liquid phase separation (LLPS) and precipitation is outlined. Appropriate solvents for precipitation have been identified by a screening approach based on solubility parameters. Feasible system compositions and solution and cloud point temperatures were determined. For the most promising solvent, cyclopentanone, a parameter study on the influence of stirring conditions during precipitation was performed. The obtained products were characterized with respect to their particle size distribution, particle shape, phase morphology, thermal properties and melt viscosity. Under optimized process conditions, PBT particles of high degree of crystallinity with narrow size distribution and a mean diameter of 36 μm were obtained; flowability was improved by dry coating. The initial PBF-LB/P processability was assessed by building single layers in a parameter study using a Formiga P110 machine. Dense single layers were obtained at laser power P L = 11.5 W, scanning speed v S = 4000 mm/s, hatch distance h S = 0.24 mm and layer thickness d S = 0.1 mm. Best to the authors' knowledge, this is the first study in the open literature reporting on a PBT feedstock material obtained by liquid-liquid phase separation and crystallization. Unlabelled Image • Development of PBT PBF feedstock with narrow size distribution, high roundness and circularity produced via precipitation. • PBT feedstock material of good flowability, high degree of crystallinity and broad PBF processing window. • Solubility parameter screening and cloud point measurement for polymer-solvent system and process condition identification. • Thorough characterization of developed PBT feedstock powder with respect to thermal, rheological and bulk solid properties. • Derivation of PBF process parameters for the precipitated PBF feedstock via parameter study on a commercial PBF machine. [ABSTRACT FROM AUTHOR]