Single-molecule fluorescence studies of intrinsically disordered proteins and liquid phase separation

Nasir, Irem; Onuchic, Paulo L.; Labra, Sergio R.; Deniz, Ashok A.

doi:10.1016/j.bbapap.2019.04.007

Cited by 35 publications

(31 citation statements)

References 85 publications

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“…Intrinsically disordered regions or proteins (IDRs or IDPs) are ubiquitous in proteomes, with ϳ25% to 30% of eukaryotic proteins being mostly disordered, and serve in a range of cellular functions, including cellular signaling, gene expression, transport, enzyme function, and flexibility and interaction promiscuity (57,58). IDP misfunction and aggregation are also associated with several diseases, including neurodegenerative diseases and cancer (59)(60)(61)(62).…”

Section: Discussionmentioning

confidence: 99%

Phase Separation of Epstein-Barr Virus EBNA2 and Its Coactivator EBNALP Controls Gene Expression

Qiu

Wang

Qin

et al. 2020

J Virol

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Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV’s transcription factors. IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.

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Section: Discussionmentioning

confidence: 99%

Phase Separation of Epstein-Barr Virus EBNA2 and Its Coactivator EBNALP Controls Gene Expression

Qiu

Wang

Qin

et al. 2020

J Virol

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“…A full description of the structures in the dense phase, the lifetime of individual interactions, and how the dynamics of one building block is correlated with that of its neighbors would open the door to a molecular understanding of the emergent material properties of dense phases. We expect that an integrative approach utilizing solution- [28,31] and solid-state NMR spectroscopy, scattering and single molecule fluorescence techniques [32] together with molecular simulations [33,34] will facilitate our understanding of how behavior at the molecular level correlates with the emergent material properties of condensates. Cryo-EM tomography [3] may play a particularly important role as it will allow bridging between structural biology of condensates in vitro and in cells.…”

Section: Multivalent Domain/motif Interactionsmentioning

confidence: 99%

Molecular structure in biomolecular condensates

Mittag

2020

Current Opinion in Structural Biology

117

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Evidence accumulated over the past decade provides support for liquid-liquid phase separation as the mechanism underlying the formation of biomolecular condensates, which include not only "membraneless" organelles such as nucleoli and RNA granules, but additional assemblies involved in transcription, translation and signaling. Understanding the molecular mechanisms of condensate function requires knowledge of the structures of their constituents. Current knowledge suggests that structures formed via multivalent domain-motif interactions remain largely unchanged within condensates. Two different viewpoints exist regarding structures of disordered low-complexity domains within condensates; one argues that low-complexity domains remain largely disordered in condensates and their multivalency is encoded in short motifs called "stickers", while the other argues that the sequences form cross-b structures resembling amyloid fibrils. We review these viewpoints and highlight outstanding questions that will inform structure-function relationships for biomolecular condensates.

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“…Characterization of single molecules and their dynamic behaviour in vivo is still challenging. Live cell confocal imaging based on single-molecule Förster resonance energy transfer (FRET) is an established tool to study biophysical features of IDPs in vitro [227,228], but was recently also proven to be a versatile tool to study biophysical features of IDPs in vivo, e.g., dimensions, submicrosecond chain dynamics or conformational changes upon interaction with binding partners [229][230][231][232]. However, plant proteins are usually imaged at the organismal level and imaging is thus based on expression of fluorescent fusion proteins, rather than on microinjected proteins that are chemically linked with fluorescent dyes.…”

Section: Methodological Advances and Outlookmentioning

confidence: 99%

“…However, plant proteins are usually imaged at the organismal level and imaging is thus based on expression of fluorescent fusion proteins, rather than on microinjected proteins that are chemically linked with fluorescent dyes. Due to weaker photophysical features of these fusion proteins, their application for extended single molecule FRET is limited [227]. Additionally, fusion proteins are bulky molecules in comparison to fluorescent dyes and might influence the accuracy of measurements.…”

Section: Methodological Advances and Outlookmentioning

confidence: 99%

Common Functions of Disordered Proteins across Evolutionary Distant Organisms

Wallmann

Kesten

2020

IJMS

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Intrinsically disordered proteins and regions typically lack a well-defined structure and thus fall outside the scope of the classic sequence-structure-function relationship. Hence, classic sequence-or structure-based bioinformatic approaches are often not well suited to identify homology or predict the function of unknown intrinsically disordered proteins. Here, we give selected examples of intrinsic disorder in plant proteins and present how protein function is shared, altered or distinct in evolutionary distant organisms. Furthermore, we explore how examining the specific role of disorder across different phyla can provide a better understanding of the common features that protein disorder contributes to the respective biological mechanism. being defined by a separate set of parameters compared to their structured and globular counterparts [7]. Hence, identification of distantly related IDPs cannot rely on structure, which can help to detect relationships of folded proteins that would remain undetected by conventional sequence-based methods [8]. Methods that use antibodies to metazoan proteins to identify putative plant homologues may result in a higher incidence of artefacts when compared to folded protein targets, due to the smaller size of disordered epitopes and their comparably higher sensitivity to epitope variation [9].In contrast to folded proteins, IDPs and IDRs show an overall increased rate of evolution, while these rates can strongly vary between different parts of the IDP [10,11]. Moreover, disordered regions appear to be more permissive to mutation, further complicating sequence-function analysis. Overall, genome duplication events seem to influence the distribution of IDRs within genomes, as the amount of identical paralogous IDRs positively correlates with the number of chromosomes [12]. Interestingly, proteins can display different modes of how sequence conservation can relate to disorder as a functional feature [13]. While in some cases only the disorder itself is conserved, but not the sequence facilitating it (flexible disorder), other IDPs retain disorder together with a highly conserved sequence (constrained disorder). Short-linear motifs (SLiMs) represent conserved functional modules that can mediate low-affinity interactions and are often interspersed by regions of flexible disorder [14]. As many alignment algorithms require long stretches of sequence similarity to satisfy statistical significance, the high modality and low complexity of SLiMs can obscure the search for homologous sequences considerably. Due to their limited size (7-12 residues), these motifs can easily develop in unrelated proteins in convergent evolution. Furthermore, post-translational modifications (PTMs) that often regulate IDP function complicate the analysis, as they can drastically alter the biophysical features of a protein and phosphorylation sites display short and weakly conserved motifs that are often difficult to detect via computational sequence analysis [15].Within protein interaction networks, IDPs ofte...

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Single-molecule fluorescence studies of intrinsically disordered proteins and liquid phase separation

Cited by 35 publications

References 85 publications

Phase Separation of Epstein-Barr Virus EBNA2 and Its Coactivator EBNALP Controls Gene Expression

Phase Separation of Epstein-Barr Virus EBNA2 and Its Coactivator EBNALP Controls Gene Expression

Molecular structure in biomolecular condensates

Common Functions of Disordered Proteins across Evolutionary Distant Organisms

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