Search

Abstract RXRβ is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes – RXRα and RXRγ – RXRβ has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRβ (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid–liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR – RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR. Video Abstract

RXRβ is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes – RXRα and RXRγ – RXRβ has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRβ (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid–liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR – RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR. 89Jx1wwVvjJQ45uAcpVb5S Video Abstract [ABSTRACT FROM AUTHOR]

As the world continues to experience the effects of SARS‐CoV‐2, there is evidence to suggest that the sequelae of viral infection (the post‐COVID‐19 condition; PCC) at both an individual and population level will be significant and long‐lasting. The history of pandemics or epidemics in the last 100 years caused by members of the RNA virus family, of which coronaviruses are a member, provides ample evidence of the acute neurological effects. However, except for the H1N1 influenza pandemic of 1918/1919 (the Spanish flu) with its associated encephalitis lethargica, there is little information on long‐term neurological sequelae. COVID‐19 is the first pandemic that has occurred in a setting of an aging population, especially in several high‐income countries. Its survivors are at the greatest risk for developing neurodegenerative conditions as they age, rendering the current pandemic a unique paradigm not previously witnessed. The SARS‐CoV‐2 virus, among the largest of the RNA viruses, is a single‐stranded RNA that encodes for 29 proteins that include the spike protein that contains the key domains required for ACE2 binding, and a complex array of nonstructural proteins (NSPs) and accessory proteins that ensure the escape of the virus from the innate immune response, allowing for its efficient replication, translation, and exocytosis as a fully functional virion. Increasingly, these proteins are also recognized as potentially contributing to biochemical and molecular processes underlying neurodegeneration. In addition to directly being taken up by brain endothelium, the virus or key protein constituents can be transported to neurons, astrocytes, and microglia by extracellular vesicles and can accelerate pathological fibril formation. The SARS‐CoV‐2 nucleocapsid protein is intrinsically disordered and can participate in liquid condensate formation, including as pathological heteropolymers with neurodegenerative disease‐associated RNA‐binding proteins such as TDP‐43, FUS, and hnRNP1A. As the SARS‐CoV‐2 virus continues to mutate under the immune pressure exerted by highly efficacious vaccines, it is evolving into a virus with greater transmissibility but less severity compared with the original strain. The potential of its lingering impact on the nervous system thus has the potential to represent an ongoing legacy of an even greater global health challenge than acute infection. [ABSTRACT FROM AUTHOR]

Simple Summary: Speckles and other nuclear bodies, the nucleolus and perinucleolar zone, transcription/replication factories and the lamina-associated compartment, serve as a structural basis for various genomic functions. In turn, genome activity and specific chromatin 3D organization directly impact the integrity of intranuclear assemblies, initiating/facilitating their formation and dictating their composition. Thus, the large-scale nucleus structure and genome activity mutually influence each other. The cell nucleus is frequently considered a compartment in which the genome is placed to protect it from external forces. Here, we discuss the evidence demonstrating that the cell nucleus should be considered, rather, as structure built around the folded genome. Decondensing chromosomes provide a scaffold for the assembly of the nuclear envelope after mitosis, whereas genome activity directs the assembly of various nuclear compartments, including nucleolus, speckles and transcription factories. The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA's genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the "box" in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses. [ABSTRACT FROM AUTHOR]

In recent years, attention has been devoted to proteins forming immiscible liquid phases within the liquid intracellular medium, commonly referred to as membraneless organelles (MLO). These organelles enable the spatiotemporal associations of cellular components that exchange dynamically with the cellular milieu.The dysregulation of these liquid–liquid phase separation processes (LLPS) may cause various diseases including neurodegenerative pathologies and cancer, among others.Until very recently, databases containing information on proteins forming MLOs, as well as tools and resources facilitating their analysis, were missing. This has recently changed with the publication of 4 databases that focus on different types of experiments, sets of proteins, inclusion criteria, and levels of annotation or curation.In this study we integrate and analyze the information across these databases, complement their records, and produce a consolidated set of proteins that enables the investigation of the LLPS phenomenon. To gain insight into the features that characterize different types of MLOs and the roles of their associated proteins, they were grouped into categories: High Confidence MLO associated (including Drivers and reviewed proteins), Potential Clients and Regulators, according to their annotated functions. We show that none of the databases taken alone covers the data sufficiently to enable meaningful analysis, validating our integration effort as essential for gaining better understanding of phase separation and laying the foundations for the discovery of new proteins potentially involved in this important cellular process.Lastly, we developed a server, enabling customized selections of different sets of proteins based on MLO location, database, disorder content, among other attributes (https://mlos.leloir.org.ar).

The cellular environment, characterized by its intricate composition and spatial organization, hosts a variety of organelles, ranging from membrane-bound ones to membraneless structures that are formed through liquid-liquid phase separation. Cells show precise control over the position of such condensates. We demonstrate that organelle movement in external concentration gradients, diffusiophoresis , is distinct from the one of colloids because fluxes can remain finite inside the liquid-phase droplets and movement of the latter arises from incompressibility. Within cellular domains diffusiophoresis naturally arises from biochemical reactions that are driven by a chemical fuel and produce waste. Simulations and analytical arguments within a minimal model of reaction-driven phase separation reveal that the directed movement stems from two contributions: Fuel and waste are refilled or extracted at the boundary, resulting in concentration gradients, which (i) induce product fluxes via incompressibility and (ii) result in an asymmetric forward reaction in the droplet's surroundings (as well as asymmetric backward reaction inside the droplet), thereby shifting the droplet's position. We show that the former contribution dominates and sets the direction of the movement, toward or away from fuel source and waste sink, depending on the product molecules' affinity toward fuel and waste, respectively. The mechanism thus provides a simple means to organize condensates with different composition. Particle-based simulations and systems with more complex reaction cycles corroborate the robustness and universality of this mechanism.

We have investigated the pressure‐ and temperature‐induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA‐binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low‐complexity domain fused with protein‐G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure‐induced and cold temperature‐induced unfolding expected for small globular domains, the low‐complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low‐complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding‐unfolding transition. Our results suggest the presence of a low‐lying extended and fully solvated state(s) of the low‐complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles. [ABSTRACT FROM AUTHOR]

The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA’s genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the “box” in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses. [Display omitted] • A splice variant of Dynamin 1, Dyn1xA, specifically mediates ultrafast endocytosis • Dyn1xA forms condensates with Syndapin 1 and pre-accumulates at endocytic zones • Syndapin 1 acts as a hub between Dyn1xA and the plasma membrane • Disruption of Dyn1xA condensates slows down the kinetics of endocytosis by 100-fold Imoto et al. demonstrate that a splice variant of Dynamin 1, Dyn1xA, mediates vesicle scission during ultrafast endocytosis. For such a rapid event, Dyn1xA molecules are concentrated at endocytic zones through molecular condensation with Syndapin 1. This cache of Dyn1xA accelerates the kinetics of endocytosis by 100-fold. [ABSTRACT FROM AUTHOR]

In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and colocalization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/or the PLD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid–liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections. [ABSTRACT FROM AUTHOR]

In recent years, attention has been devoted to proteins forming immiscible liquid phases within the liquid intracellular medium, commonly referred to as membraneless organelles (MLO). These organelles enable the spatiotemporal associations of cellular components that exchange dynamically with the cellular milieu. The dysregulation of these liquid-liquid phase separation processes (LLPS) may cause various diseases including neurodegenerative pathologies and cancer, among others. Until very recently, databases containing information on proteins forming MLOs, as well as tools and resources facilitating their analysis, were missing. This has recently changed with the publication of 4 databases that focus on different types of experiments, sets of proteins, inclusion criteria, and levels of annotation or curation. In this study we integrate and analyze the information across these databases, complement their records, and produce a consolidated set of proteins that enables the investigation of the LLPS phenomenon. To gain insight into the features that characterize different types of MLOs and the roles of their associated proteins, they were grouped into categories: High Confidence MLO associated (including Drivers and reviewed proteins), Potential Clients and Regulators, according to their annotated functions. We show that none of the databases taken alone covers the data sufficiently to enable meaningful analysis, validating our integration effort as essential for gaining better understanding of phase separation and laying the foundations for the discovery of new proteins potentially involved in this important cellular process. Lastly, we developed a server, enabling customized selections of different sets of proteins based on MLO location, database, disorder content, among other attributes (https://forti.shinyapps.io/mlos/)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)