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.     

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.     

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.     

Phase-Separated Subcellular Compartmentation and Related Human Diseases

In live cells, proteins and nucleic acids can associate together through multivalent interactions, and form relatively isolated phases that undertake designated biological functions and activities. In the past decade, liquid–liquid phase separation (LLPS) has gradually been recognized as a general mechanism for the intracellular organization of biomolecules. LLPS regulates the assembly and composition of dozens of membraneless organelles and condensates in cells. Due to the altered physiological conditions or genetic mutations, phase-separated condensates may undergo aberrant formation, maturation or gelation that contributes to the onset and progression of various diseases, including neurodegenerative disorders and cancers. In this review, we summarize the properties of different membraneless organelles and condensates, and discuss multiple phase separation-regulated biological processes. Based on the dysregulation and mutations of several key regulatory proteins and signaling pathways, we also exemplify how aberrantly regulated LLPS may contribute to human diseases.     

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.     

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.     

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.