Liquid–Liquid Phase Separation in the Presence of Macromolecular Crowding and State-dependent Kinetics

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.     

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.     

BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid–Liquid Phase Separation

The liquid–liquid phase separation (LLPS) of biomolecules is a phenomenon which is nowadays recognized as the driving force for the biogenesis of numerous functional membraneless organelles and cellular bodies. The interplay between the protein primary sequence and phase separation remains poorly understood, despite intensive research. To uncover the sequence-encoded signals of protein capable of undergoing LLPS, we developed a novel web platform named BIAPSS (Bioinformatics Analysis of LLPS Sequences). This web server provides on-the-fly analysis, visualization, and interpretation of the physicochemical and structural features for the superset of curated LLPS proteins.     

Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1–2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40–50 mOsm). Both reassembled into new structures within 1–2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90–175 mOsm) first rapidly disassembled within 1–3 min, and then, in most cells, spontaneously reassembled 7–15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.     

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

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.     

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML^−/−), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies.     

Different Intermolecular Interactions Drive Nonpathogenic Liquid–Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43

The liquid–liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix–helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix–helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix–helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.     

The Patterning and Proportion of Charged Residues in the Arginine-Rich Mixed-Charge Domain Determine the Membrane-Less Organelle Targeted by the Protein

Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.     

Tau N-Terminal Inserts Regulate Tau Liquid-Liquid Phase Separation and Condensates Maturation in a Neuronal Cell Model

The microtubule-associated protein tau can undergo liquid–liquid phase separation (LLPS) to form membraneless condensates in neurons, yet the underlying molecular mechanisms and functions of tau LLPS and tau droplets remain to be elucidated. The human brain contains mainly 6 tau isoforms with different numbers of microtubule-binding repeats (3R, 4R) and N-terminal inserts (0N, 1N, 2N). However, little is known about the role of N-terminal inserts. Here we observed the dynamics of three tau isoforms with different N-terminal inserts in live neuronal cell line HT22. We validated tau LLPS in cytoplasm and found that 2N-tau forms liquid-like, hollow-shell droplets. Tau condensates became smaller in 1N-tau comparing with 2N-tau, while no obvious tau accumulated dots were shown in 0N-tau. The absence of N-terminal inserts significantly affected condensate colocalization of tau and p62. The results reveal insights into the tau LLPS assembly mechanism and functional effects of N-terminal inserts in tau.     

The Multivalent Polyampholyte Domain of Nst1, a P-Body-Associated Saccharomyces cerevisiae Protein,Provides a Platform for Interacting with P-Body Components

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1_∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1_∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1_∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components.     

Recent Findings Related to Cardiomyopathy and Genetics

With the development and advancement of next-generation sequencing (NGS), genetic analysis is becoming more accessible. High-throughput genetic studies using NGS have contributed to unraveling the association between cardiomyopathy and genetic background, as is the case with many other diseases. Rare variants have been shown to play major roles in the pathogenesis of cardiomyopathy, which was empirically recognized as a monogenic disease, and it has been elucidated that the clinical course of cardiomyopathy varies depending on the causative genes. These findings were not limited to dilated and hypertrophic cardiomyopathy; similar trends were reported one after another for peripartum cardiomyopathy (PPCM), cancer therapy-related cardiac dysfunction (CTRCD), and alcoholic cardiomyopathy (ACM). In addition, as the association between clinical phenotypes and the causative genes becomes clearer, progress is being made in elucidating the mechanisms and developing novel therapeutic agents. Recently, it has been suggested that not only rare variants but also common variants contribute to the development of cardiomyopathy. Cardiomyopathy and genetics are approaching a new era, which is summarized here in this overview.     

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine—Chromatographic Studies

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography–mass spectrometry (GC–MS) based method was elaborated to identify and quantify TCA in human urine. The GC–MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1–50 µmol L⁻¹ range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.     

Nst1, Densely Associated to P-Body in the Post-Exponential Phases of Saccharomyces cerevisiae,Shows an Intrinsic Potential of Producing Liquid-Like Condensates of P-Body Components in Cells

Membrane-less biomolecular compartmentalization is a core phenomenon involved in many physiological activities that occur ubiquitously in cells. Condensates, such as promyelocytic leukemia (PML) bodies, stress granules, and P-bodies (PBs), have been investigated to understand the process of membrane-less cellular compartmentalization. In budding yeast, PBs dispersed in the cytoplasm of exponentially growing cells rapidly accumulate in response to various stresses such as osmotic stress, glucose deficiency, and heat stress. In addition, cells start to accumulate PBs chronically in post-exponential phases. Specific protein–protein interactions are involved in accelerating PB accumulation in each circumstance, and discovering the regulatory mechanism for each is the key to understanding cellular condensation. Here, we demonstrate that Nst1 of budding yeast Saccharomyces cerevisiae is far more densely associated with PBs in post-exponentially growing phases from the diauxic shift to the stationary phase than during glucose deprivation of exponentially growing cells, while the PB marker Dcp2 exhibits a similar degree of condensation under these conditions. Similar to Edc3, ectopic Nst1 overexpression induces self-condensation and the condensation of other PB components, such as Dcp2 and Dhh1, which exhibit liquid-like properties. Altogether, these results suggest that Nst1 has the intrinsic potential for self-condensation and the condensation of other PB components, specifically in post-exponential phases.     

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.     

Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases

Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood–brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer’s disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood–brain barrier and neurodegenerative diseases.