Macromolecular crowding sensing during osmotic stress in plants.

Osmotic stress conditions occur at multiple stages of plant life. Changes in water availability caused by osmotic stress induce alterations in the mechanical properties of the plasma membrane, its interaction with the cell wall, and the concentration of macromolecules in the cytoplasm. We summarize the reported players involved in the sensing mechanisms of osmotic stress in plants. We discuss how changes in macromolecular crowding are perceived intracellularly by intrinsically disordered regions (IDRs) in proteins. Finally, we review methods for dynamically monitoring macromolecular crowding in living cells and discuss why their implementation is required for the discovery of new plant osmosensors. Elucidating the osmosensing mechanisms will be essential for designing strategies to improve plant productivity in the face of climate change.

Beyond RNA binding domains: determinants of protein-RNA binding.

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

Transcriptional enhancers at 40: evolution of a viral DNA element to nuclear architectural structures.

Gene regulation by transcriptional enhancers is the dominant mechanism driving cell type- and signal-specific transcriptional diversity in metazoans. However, over four decades since the original discovery, how enhancers operate in the nuclear space remains largely enigmatic. Recent multidisciplinary efforts combining real-time imaging, genome sequencing, and biophysical strategies provide insightful but conflicting models of enhancer-mediated gene control. Here, we review the discovery and progress in enhancer biology, emphasizing the recent findings that acutely activated enhancers assemble regulatory machinery as mesoscale architectural structures with distinct physical properties. These findings help formulate novel models that explain several mysterious features of the assembly of transcriptional enhancers and the mechanisms of spatial control of gene expression.

RNA-dependent proteome solubility maintenance in Escherichia coli lysates analysed by quantitative mass spectrometry: Proteomic characterization in terms of isoelectric point, structural disorder, functional hub, and chaperone network.

Protein aggregation, a consequence of misfolding and impaired proteostasis, can lead to cellular malfunctions such as various proteinopathies. The mechanisms protecting proteins from aggregation in complex cellular environments have long been investigated, often from a protein-centric viewpoint. However, our study provides insights into a crucial, yet overlooked actor: RNA. We found that depleting RNAs from Escherichia coli lysates induces global protein aggregation. Our quantitative mass spectrometry analysis identified over 900 statistically significant proteins from the Escherichia coli proteome whose solubility depends on RNAs. Proteome-wide characterization showed that the RNA dependency is particularly enriched among acidic proteins, intrinsically disordered proteins, and structural hub proteins. Moreover, we observed distinct differences in RNA-binding mode and Gene Ontology categories between RNA-dependent acidic and basic proteins. Notably, the solubility of key molecular chaperones [Trigger factor, DnaJ, and GroES] is largely dependent on RNAs, suggesting a yet-to-be-explored hierarchical relationship between RNA-based chaperone (termed as chaperna) and protein-based chaperones, both of which constitute the whole chaperone network. These findings provide new insights into the RNA-centric role in maintaining healthy proteome solubility in vivo, where proteins associate with a variety of RNAs, either stably or transiently.

Protein intrinsically disordered region prediction by combining neural architecture search and multi-objective genetic algorithm.

BACKGROUND: Intrinsically disordered regions (IDRs) are widely distributed in proteins and related to many important biological functions. Accurately identifying IDRs is of great significance for protein structure and function analysis. Because the long disordered regions (LDRs) and short disordered regions (SDRs) share different characteristics, the existing predictors fail to achieve better and more stable performance on datasets with different ratios between LDRs and SDRs. There are two main reasons. First, the existing predictors construct network structures based on their own experiences such as convolutional neural network (CNN) which is used to extract the feature of neighboring residues in protein, and long short-term memory (LSTM) is used to extract the long-distance dependencies feature of protein residues. But these networks cannot capture the hidden feature associated with the length-dependent between residues. Second, many algorithms based on deep learning have been proposed but the complementarity of the existing predictors is not fully explored and used. RESULTS: In this study, the neural architecture search (NAS) algorithm was employed to automatically construct the network structures so as to capture the hidden features in protein sequences. In order to stably predict both the LDRs and SDRs, the model constructed by NAS was combined with length-dependent models for capturing the unique features of SDRs or LDRs and general models for capturing the common features between LDRs and SDRs. A new predictor called IDP-Fusion was proposed. CONCLUSIONS: Experimental results showed that IDP-Fusion can achieve more stable performance than the other existing predictors on independent test sets with different ratios between SDRs and LDRs.

Intrinsically disordered proteins and liquid-liquid phase separation in SARS-CoV-2 interactomes.

This paper discusses the properties of proteins and their relations in the interactomes of the selected subsets of SARS-CoV-2 proteome-the membrane protein, nonstructural proteins, and, finally, full proteome. Protein disorder according to several measures, liquid-liquid phase separation probabilities, and protein node degrees in the interaction networks were singled out as the features of interest. Additionally, viral interactomes were combined with the interactome of human lung tissue so as to examine if the new connections in the resulting viral-host interactome are linked to protein disorder. Correlation analysis shows that there is no clear relationship between raw features of interest, whereas there is a positive correlation between the protein disorder and its neighborhood mean disorder. There are also indications that highly connected viral hubs tend to be on average more ordered than proteins with a small number of connections. This is in contrast to previous similar studies conducted on eukaryotic interactomes and possibly raises new questions in research on viral interactomes.

Insights into the Cellular Localization and Functional Properties of TSPYL5 Protein.

In recent years, the role of liquid-liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4-27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198-382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1-198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid-liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell's molecular life.

Identification of intrinsically disordered regions in hub genes of acute myeloid leukemia: A bioinformatics approach.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Over the past decades, there has been a great challenge in the treatment of AML. A combination of gene expression profiling with computational approaches can lead to the identification of hub genes in AML. However, it is important to study the structure of these hub genes considering their importance in the protein-protein interaction (PPI) network of specific cancer. In this study, we designed an integrated method to analyze the presence of intrinsically disordered regions (IDRs) in selected hub genes of AML. A gene expression profile of AML was obtained from Gene Expression Omnibus (GEO) database. Further analysis identified differentially expressed genes (DEGs) in AML. Additionally, the top 15 hub genes following construction and analysis of the PPI network of DEGs were selected. Validation of hub genes revealed that there is a reverse relationship between overexpression of FLT3, PPBP, and PF4 genes and the survival of AML patients. Based on IDRs investigation, FLT3 and PF4 are partially disordered, while PPBP is mostly disordered. Through clustering the network into structural modules, we identified two important modules in the PPI network of DEGs that showed the important position of PPBP in module 1. Based on further analysis of protein flexibility and its important role in biological processes, we suggest that PPBP can be considered as a potential drug target in AML.

RNA-Binding Proteins as Epigenetic Regulators of Brain Functions and Their Involvement in Neurodegeneration.

A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and translation of the transcripts. Notably, most RBPs contain intrinsically disordered regions (IDRs) that are thought to be involved in the formation of membrane-less structures, probably due to liquid-liquid phase separation (LLPS). Such structures are evidenced as a variety of granules that contain proteins and different classes of RNAs. The other side of the peculiar properties of IDRs is, however, that, under altered cellular conditions, they are also prone to form aggregates, as observed in neurodegeneration. Interestingly, RBPs, as part of both normal and aggregated complexes, are also able to enter extracellular vesicles (EVs), and in doing so, they can also reach cells other than those that produced them.

Molecular insights into the interaction between a disordered protein and a folded RNA.

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.

Interplay between posttranslational modifications and liquid‒liquid phase separation in tumors.

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.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome-Proteome Interface.

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells.

Analysis of Phase-Separated Biomolecular Condensates in Cancer.

Phase-separated biomolecular condensates play important roles in virtually all cellular processes, and their dysregulation is associated with many pathological processes including cancer. Here we concisely review some basic methodologies and strategies to analyze the phase-separated biomolecular condensates in cancer, including physical characterization of phase separation for the protein of interest, functional demonstration of this property in cancer regulation, as well as mechanistic studies on how phase separation regulates the protein's function in cancer.

Through the lens of phase separation: intrinsically unstructured protein and chromatin looping.

The establishment, maintenance and dynamic regulation of three-dimensional (3D) chromatin structures provide an important means for partitioning of genome into functionally distinctive domains, which helps to define specialized gene expression programs associated with developmental stages and cell types. Increasing evidence supports critical roles for intrinsically disordered regions (IDRs) harbored within transcription factors (TFs) and chromatin-modulatory proteins in inducing phase separation, a phenomenon of forming membrane-less condensates through partitioning of biomolecules. Such a process is also critically involved in the establishment of high-order chromatin structures and looping. IDR- and phase separation-driven 3D genome (re)organization often goes wrong in disease such as cancer. This review discusses about recent advances in understanding how phase separation of intrinsically disordered proteins (IDPs) modulates chromatin looping and gene expression.

How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization.

The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila PhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker.

Comparing the effects of proteins with IDRs on membrane system in yeast, mammalian cells, and the model plant Arabidopsis.

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.

Ocular disease-associated mutations diminish the mitotic chromosome retention ability of PAX6.

Transcription factors play a key role in maintaining cell identity. One mechanism of such cell memory after multiple rounds of cell division cycles is through persistent mitotic chromosome binding, although how individual transcription factors achieve mitotic chromosome retention is not completely understood. Here we show that PAX6, a lineage-determining transcription factor, coats mitotic chromosomes. Using deletion and point mutants associated with human ocular diseases in live-cell imaging analysis, we identified two regions, MCR-D1 and MCR-D2, that were responsible for mitotic chromosome retention of PAX6. We also identified three nuclear localization signals (NLSs) that contributed to mitotic chromosome retention independent of their nuclear import functions. Full mitotic chromosome retention required the presence of DNA-binding domains as well as NLSs within MCR-Ds. Furthermore, disease-associated mutations and NLS mutations changed the distribution of intrinsically disordered regions (IDRs) in PAX6. Our findings not only identify PAX6 as a novel mitotic chromosome retention factor but also demonstrate that the mechanism of mitotic chromosome retention involves sequence-specific DNA binding, NLSs, and molecular conformation determined by IDRs. These findings link mitotic chromosome retention with PAX6-related pathogenesis and imply similar mechanisms for other lineage-determining factors in the PAX family.

Intrinsically disordered regions are abundant in simplexvirus proteomes and display signatures of positive selection.

Whereas the majority of herpesviruses co-speciated with their mammalian hosts, human herpes simplex virus 2 (HSV-2, genus Simplexvirus) most likely originated from the cross-species transmission of chimpanzee herpesvirus 1 to an ancestor of modern humans. We exploited the peculiar evolutionary history of HSV-2 to investigate the selective events that drove herpesvirus adaptation to a new host. We show that HSV-2 intrinsically disordered regions (IDRs)-that is, protein domains that do not adopt compact three-dimensional structures-are strongly enriched in positive selection signals. Analysis of viral proteomes indicated that a significantly higher portion of simplexvirus proteins is disordered compared with the proteins of other human herpesviruses. IDR abundance in simplexvirus proteomes was not a consequence of the base composition of their genomes (high G + C content). Conversely, protein function determines the IDR fraction, which is significantly higher in viral proteins that interact with human factors. We also found that the average extent of disorder in herpesvirus proteins tends to parallel that of their human interactors. These data suggest that viruses that interact with fast-evolving, disordered human proteins, in turn, evolve disordered viral interactors poised for innovation. We propose that the high IDR fraction present in simplexvirus proteomes contributes to their wider host range compared with other herpesviruses.

Intrinsically Disordered Human T Lymphotropic Virus Type 1 p30 Protein: Experimental and Computational Evidence.

Human T lymphotropic virus type 1 (HTLV-1) causes adult T cell leukemia and lymphoma and other neuroinflammatory diseases. The pX region of HTLV-1 genome encodes an accessory protein p30 that is required for viral persistence and spread in the host. p30 regulates viral gene expression at the transcription level by competing with Tax for p300 binding and at posttranscriptional level by nuclear retention of tax/rex messenger RNA (mRNA). In addition, p30 modulates the host cellular environment by binding to various host proteins such as ATM, REGγ, and PRMT5. However, the low expression levels of p30 has been a major hurdle in studying its structure-function relationship in the context of HTLV-1 pathobiology, which is most likely due to its intrinsically disordered nature. To investigate the unstable nature of p30, flow cytometric analysis of p30-GFP fusion protein expressed in Escherichia coli was conducted and bioinformatics analysis of p30 was performed. The bacterial cells were green fluorescent protein (GFP) positive, indicating that p30-GFP was in the soluble fraction. Induction, particularly at higher temperature, reduced the expression of p30-GFP. Moreover, p30-GFP was detected exclusively in insoluble fraction upon cell lysis, suggesting its unstable and disordered nature. The bioinformatics analysis of p30 protein sequence and amino acid content revealed that p30 has highly disordered regions from amino acids 75-155 and 197-241. Furthermore, p30 has regions for macromolecular interactions that could stabilize it and these regions coincide with the unstable regions. Collectively, the study indicates that HTLV-1 p30 is an intrinsically disordered protein.