Low-complexity Domain Research Articles

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

Thermodynamic Stability of hnRNP A1 Low Complexity Domain Revealed by High-Pressure NMR

Low-complexity (LC) domains found in RNA-binding proteins associated with liquid-liquid phase separation form a specific class of intrinsically disordered domains. LC domains have distinct amino acid compositions; they are enriched in polar amino acids (especially Ser and Gly), and feature conserved patterns of aromatic residues. in many cases, these disordered LC domains are necessary and sufficient for driving phase separation. Liquid condensates are believed to be the result of multivalent weak interactions between multiple interacting motifs in LC domains, including electrostatic, cation-pi and pi-pi interactions.

Hidden Complexities in the Phase Behavior of Low-Complexity Disordered Proteins

Liquid-liquid phase separation (LLPS) is the underlying driving force for membrane-less compartmentalization in cells. LLPS plays key roles in fundamental cell biological processes and is often mediated by disordered low-complexity domains (LCDs). How the driving forces for LLPS are encoded in LCDs and how the phase behavior can be predicted from their sequence is incompletely understood. We are using the stickers-and-spacers framework to reconstruct the full phase behavior. Stickers form non-covalent inter- and intramolecular crosslinks, whereas spacers either enable or suppress the formation of these crosslinks. We have previously shown that aromatic residues are the stickers in the polar prion-like LCD of hnRNPA1. However, the balance of tyrosine and phenylalanine residues varies across LCDs and charged residues may contribute to LLPS in sequences with higher charge content. Herein we (i) extend the stickers-and-spacers model to account for interactions of charged residues and (ii) disentangle the interaction strength of phenylalanine and tyrosine for themselves and for each other. We demonstrate that tyrosine is a stronger sticker than phenylalanine and negatively charged residues are solubilizing spacers. Arginines act as stickers through pairwise interactions with aromatic residues. Importantly, low or high values of the net charge per residue weaken the driving force for LLPS via mean-field electrostatic effects. We further demonstrate that the phase behavior correlates well with the single-chain dimensions of LCD variants with different fractions of aromatic residues, but that this correlation can be broken by mean-field electrostatic effects. Our results support that the stickers-and-spacers model can be adapted for LCDs with a wide range of sequence characteristics, promising precise prediction of phase behavior from the sequence alone.

Exploring the Effects of Increased Charge Content on the Phase Behavior of a Protein Low Complexity Domain

Exploring the Effects of Increased Charge Content on the Phase Behavior of a Protein Low Complexity Domain

Characterization of the Structural Features that Mediate Phase Separation of Intrinsically Disordered Low-Complexity Domains

Recently, much insight has been gained regarding the sequence features of intrinsically disordered low complexity domains that drive the formation of biomolecular condensates. However, there is still controversy regarding what structural features drive condensate formation. One school of thought posits that transient interactions are formed between sticky side chains in an otherwise disordered chain, even inside of dense phases. The other side argues that interactions between short motifs that form cross-β structure drives phase separation.

How do Evolutionarily Encoded Compositional Biases Influence the Phase Behaviors of Intrinsically Disordered Low Complexity Domains of Proteins?

How do Evolutionarily Encoded Compositional Biases Influence the Phase Behaviors of Intrinsically Disordered Low Complexity Domains of Proteins?

Real-Time Observation of Structural and Dynamical Changes of the FUS Low-Complexity Domain During Liquid-To-Solid Phase Transitions

Real-Time Observation of Structural and Dynamical Changes of the FUS Low-Complexity Domain During Liquid-To-Solid Phase Transitions

The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 μM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.

A generic approach to study the kinetics of liquid\u2013liquid phase separation under near-native conditions

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation.

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Driving force of biomolecular liquid–liquid phase separation probed by nuclear magnetic resonance spectroscopy

The assembly of biomolecular condensates is driven by liquid–liquid phase separation. To understand the structure and functions of these condensates, it is essential to characterize the underlying driving forces, <i>e</i>.<i>g</i>., protein–protein and protein–RNA interactions. As both structured and low-complexity domains are involved in the phase separation process, NMR is probably the only technique that can be used to depict the binding topology and interaction modes for the structured and nonstructured domains simultaneously. Atomic-resolution analysis for the intramolecular and intermolecular interactions between any pair of components sheds light on the mechanism for phase separation and biomolecular condensate assembly and disassembly. Herein, we describe the procedures used for the most extensively employed NMR techniques to characterize key interactions for biomolecular phase separation.

Regulation of processing bodies: From viruses to cancer epigenetic machinery.

Processing bodies (PBs) are 100-300 nm cytoplasmic messenger ribonucleoprotein particle (mRNP) granules that regulate eukaryotic gene expression. These cytoplasmic compartments harbor messenger RNAs (mRNAs) and several proteins involved in mRNA decay, microRNA silencing, nonsense-mediated mRNA decay, and splicing. Though membrane-less, PB structures are maintained by RNA-protein and protein-protein interactions. PB proteins have intrinsically disordered regions and low complexity domains, which account for its liquid to liquid phase separation. In addition to being dynamic and actively involved in the exchange of materials with other mRNPs and organelles, they undergo changes on various cellular cues and environmental stresses, including viral infections. Interestingly, several PB proteins are individually implicated in cancer development, and no study has addressed the effects on PB dynamics after epigenetic modifications of cancer-associated PB genes. In the current review, we summarize modulations undergone by P bodies or P body components upon viral infections. Furthermore, we discuss the selective and widely investigated PB proteins that undergo methylation changes in cancer and their potential as biomarkers.

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Simulation of FUS Protein Condensates with an Adapted Coarse-Grained Model.

Disordered proteins and nucleic acids can condense into droplets that resemble the membraneless organelles observed in living cells. MD simulations offer a unique tool to characterize the molecular interactions governing the formation of these biomolecular condensates, their physicochemical properties, and the factors controlling their composition and size. However, biopolymer condensation depends sensitively on the balance between different energetic and entropic contributions. Here, we develop a general strategy to fine-tune the potential energy function for molecular dynamics simulations of biopolymer phase separation. We rebalance protein–protein interactions against solvation and entropic contributions to match the excess free energy of transferring proteins between dilute solution and condensate. We illustrate this formalism by simulating liquid droplet formation of the FUS low-complexity domain (LCD) with a rebalanced MARTINI model. By scaling the strength of the nonbonded interactions in the coarse-grained MARTINI potential energy function, we map out a phase diagram in the plane of protein concentration and interaction strength. Above a critical scaling factor of αc ≈ 0.6, FUS-LCD condensation is observed, where α = 1 and 0 correspond to full and repulsive interactions in the MARTINI model. For a scaling factor α = 0.65, we recover experimental densities of the dilute and dense phases, and thus the excess protein transfer free energy into the droplet and the saturation concentration where FUS-LCD condenses. In the region of phase separation, we simulate FUS-LCD droplets of four different sizes in stable equilibrium with the dilute phase and slabs of condensed FUS-LCD for tens of microseconds, and over one millisecond in aggregate. We determine surface tensions in the range of 0.01–0.4 mN/m from the fluctuations of the droplet shape and from the capillary-wave-like broadening of the interface between the two phases. From the dynamics of the protein end-to-end distance, we estimate shear viscosities from 0.001 to 0.02 Pa s for the FUS-LCD droplets with scaling factors α in the range of 0.625–0.75, where we observe liquid droplets. Significant hydration of the interior of the droplets keeps the proteins mobile and the droplets fluid.

Molecular Details of Protein Condensates Probed by Microsecond Long Atomistic Simulations.

The formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS) and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Investigating the nature of these interactions, and their effect on dynamics within the condensed phase, is therefore of critical importance but very challenging for either simulation or experiment. Here, we study these interactions and their dynamics by pairing a novel multiscale simulation strategy with microsecond all-atom MD simulations of a condensed, IDP-rich phase. We simulate two IDPs this way, the low complexity domain of FUS and the N-terminal disordered domain of LAF-1, and find good agreement with experimental information about average density, water content, and residue-residue contacts. We go significantly beyond what is known from experiments by showing that ion partitioning within the condensed phase is largely driven by the charge distribution of the proteins and-in the cases considered-shows little evidence of preferential interactions of the ions with the proteins. Furthermore, we can probe the microscopic diffusive dynamics within the condensed phase, showing that water and ions are in dynamic equilibrium between dense and dilute phases, and their diffusion is reduced in the dense phase. Despite their high concentration in the condensate, the protein molecules also remain mobile, explaining the observed liquid-like properties of this phase. We finally show that IDP self-association is driven by a combination of nonspecific hydrophobic interactions as well as hydrogen bonds, salt bridges, and π-π and cation-π interactions. The simulation approach presented here allows the structural and dynamical properties of biomolecular condensates to be studied in microscopic detail and is generally applicable to single- and multicomponent systems of proteins and nucleic acids involved in LLPS.

The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-β2 (Kapβ2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapβ2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapβ2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.

Abstract PR06: Spatial sequestration of GSKα as a cellular response to amino acid starvation

Abstract The ability of cells to tolerate amino acid starvation is fundamental to survival under stress. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. We recently found that Wnt pathway activation induces a profound therapeutic vulnerability to asparaginase in acute leukemia and in colorectal cancer (Hinze et al., In press; Hinze et al., 2019). This effect is mediated by a β-catenin independent branch of Wnt signaling termed Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein degradation. Asparaginase resistance is uniquely dependent on GSK3α because its inhibition fully phenocopied Wnt-induced sensitization to asparaginase (p &amp;lt; 0.0001), whereas selective GSK3β inhibition had no effect (p=ns). This is surprising because GSK3α and GSK3β are closely related paralogs thought to be redundant for many of their biologic functions. Thus, we set out to define why asparaginase resistance is selectively dependent on GSK3α activity. To define the GSK3α domains responsible for asparaginase resistance, we tested whether resistance could be restored by expression of a series of GSK3 alleles in which the N-terminal, kinase, and C-terminal domains of GSK3α and GSK3β were swapped in various configurations. Fusing the N-terminus of GSK3α to the kinase and C-terminal domains of GSK3β fully restored asparaginase resistance in GSK3α depleted T-ALL (p&amp;lt;0.0001). By contrast, fusing the N-terminus of GSK3β to the kinase and C-terminus of GSK3α had no activity (p=ns). To investigate how the N-terminus of GSK3α regulates asparaginase response, we first applied structural prediction algorithms, which revealed that the N-terminal domain of GSK3α is a low-complexity domain, a feature lacking in its paralog GSK3β. Immunofluorescence confocal microscopy revealed that this domain mediates spatial sequestration of GSK3α into membrane-less cytoplasmic bodies in response to amino acid starvation. GSK3a bodies appeared to be distinct from known phase-separated compartments such as stress granules and aggresomes. However, GSK3α bodies colocalized with K48-linked ubiquitin and the proteasome, suggesting that they assemble the cellular machinery for protein ubiquitination and proteasomal degradation. This suggests that GSK3α body formation promotes efficient generation of amino acids via catabolic protein degradation, and promotes survival during starvation in normal and malignant cells. Importantly, GSK3α body formation was stimulated in response to starvation of several amino acids (p&amp;lt;0.0001), and promoted survival of normal cells in response to amino acid starvation. In human cancer, GSK3α body formation predicted response to asparaginase resistance (p&amp;lt;0.0001), and sensitivity to asparaginase combined with a GSK3a inhibitor (p&amp;lt;0.0001). We propose that GSK3α body formation provides a cellular mechanism to promote catalytic efficiency of protein degradation as an adaptive response to amino acid starvation. Citation Format: Laura Hinze, Sabine Schreek, Andre Zeug, Evgeni Ponimaskin, Roxane Labrosse, James Degar, Beat Bornhauser, Jean-Pierre Bourquin, Martin Stanulla, Alejandro Gutierrez. Spatial sequestration of GSKα as a cellular response to amino acid starvation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR06.

Mitigating ageing bias in article level metrics using citation network analysis

Abstract Article level scientometric indicators (ALMs) are usually of cumulative nature making articles of different age hard to compare. Here, we introduce a new ALM, the Time Debiased Significance Score (TDSS), which measures the significance of a publication based on the structure of the whole citation network and eliminates the global ageing bias in the network: older publications should not be a priori privileged or disadvantaged compared to newer ones. The TDSS is based on a modified variant of the PageRank measure, incorporating a mathematically consistent temporal detrending and ensuring a few key features: (i) the TDSS should not show any global trend as a function of the topological index (causal order in the citation network); (ii) the TDSS value of a publication should decrease as time passes (and the citation network grows) if no more citations are associated with it. The above definition is beneficial in multiple ways, including e.g. low computational complexity and weak domain dependence. Further, estimation of reliability of the TDSS and its extension to groups of items like overall score of a research group are also possible.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

Coiled-Coil Motifs of RNA-Binding Proteins: Dynamicity in RNA Regulation

Neuronal granules are biomolecular condensates that concentrate high quantities of RNAs and RNA-related proteins within neurons. These dense packets of information are trafficked from the soma to distal sites rich in polysomes, where local protein synthesis can occur. Movement of neuronal granules to distal sites, and local protein synthesis, play a critical role in synaptic plasticity. The formation of neuronal granules is intriguing; these granules lack a membrane and instead phase separate due to protein and RNA interactions. Low complexity motifs and RNA binding domains are highly prevalent in these proteins. Here, we introduce the role that coiled-coil motifs play in neuronal granule proteins, and investigate the structure-function relationship of coiled-coil proteins in RNA regulation. Interestingly, low complexity domains and coiled-coil motifs are highly dynamic, allowing for increased functional response to environmental influences. Finally, biomolecular condensates have been suggested to drive the formation of toxic, neurodegenerative proteins such as TDP-43 and tau. Here, we review the conversion of coiled-coil motifs to amyloid structures, and speculate a role that neuronal granules play in coiled-coil to amyloid conversions of neurodegenerative proteins.