scholarly journals Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants

2021 ◽  
Vol 118 (51) ◽  
pp. e2109967118
Author(s):  
Fleurie M. Kelley ◽  
Bruna Favetta ◽  
Roshan Mammen Regy ◽  
Jeetain Mittal ◽  
Benjamin S. Schuster
Keyword(s):  
Ribosome Biogenesis ◽  
Biological Function ◽  
Surface Film ◽  
Glutathione S Transferase ◽  
Intrinsically Disordered ◽  
Liquid Phases ◽  
Film Layer ◽  
And Function ◽  
Liquid Liquid Phase Separation

Cells contain membraneless compartments that assemble due to liquid–liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non–phase-separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

scholarly journals Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants

2021 ◽  
Author(s):  
Fleurie M Kelley ◽  
Bruna Favetta ◽  
Roshan M Regy ◽  
Jeetain Mittal ◽  
Benjamin S Schuster
Keyword(s):  
Ribosome Biogenesis ◽  
Surface Film ◽  
Glutathione S Transferase ◽  
Intrinsically Disordered ◽  
Liquid Phases ◽  
Film Layer ◽  
And Function ◽  
And Control ◽  
Liquid Liquid Phase Separation

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals A new class of disordered elements controls DNA replication through initiator self-assembly

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Matthew W Parker ◽  
Maren Bell ◽  
Mustafa Mir ◽  
Jonchee A Kao ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Dna Replication ◽  
Self Assembly ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Linear Arrangement ◽  
New Class ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Liquid Liquid Phase Separation

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

scholarly journals Polycomb Cbx2 Condensates Assemble through Phase Separation

2018 ◽  
Author(s):  
Roubina Tatavosian ◽  
Samantha Kent ◽  
Kyle Brown ◽  
Tingting Yao ◽  
Huy Nguyen Duc ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Group ◽  
Developmental Defects ◽  
Intrinsically Disordered ◽  
Condensate Formation ◽  
Development And Differentiation ◽  
Polycomb Repressive Complex 1 ◽  
Liquid Liquid Phase Separation

AbstractPolycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

scholarly journals Forgotten partners and function regulators of inducible metallothioneins

2019 ◽  
Vol 70 (4) ◽  
pp. 256-264
Author(s):  
Mirela Pavić ◽  
Petra Turčić ◽  
Marija Ljubojević
Keyword(s):  
Oxidative Stress ◽  
Plasma Proteins ◽  
Biological Function ◽  
Life Forms ◽  
Ros Generation ◽  
Transitional Metals ◽  
Almost All ◽  
And Function ◽  
Sex And Age Differences

AbstractMetallothioneins are peculiar cysteine rich, heat resistant, small cellular plasma proteins expressed through almost all life forms. The currently established biological functions of metallothioneins are the homeostasis of essential metals and protection against toxic transitional metals (TM) alongside defence from oxidative stress by direct scavenging of reactive oxygen and nitrogen species (ROS and RNS). In mammals, among the four main evolutionary conserved forms, only the ubiquitously expressed metallothionein 1 and 2 (here abbreviated as MT) are inducible by TM, oxidative stress, glucocorticoids and starvation among various other stimuli. However, more than sixty years after being discovered, metallothioneins still bear unresolved issues about their possible physiological function and regulation. The biological function of MTs has still not been associated with the in vitro-demonstrated capacity of MT interaction with cellular molecules glutathione (GSH) or adenosine triphosphate (ATP), or with the possibility of direct iron-MT binding in the reducing intracellular environment of some organelles, e.g. lysosomes. Iron as the most abundant cellular TM is also one of the main physiological sources of ROS. Moreover, iron exhibits strain, sex and age differences that reflected ROS generation and MT induction in (patho)physiology and toxicology studies. A recent study showed that iron sex differences follows expression of both ferritin and MT leading to wide implications from essential TM interconnectivity to aging. This review places emphasis on biochemically proven but physiologically ignored interactions of MT with iron to stimulate advanced research for establishing a wide frame of the biological roles of MTs important for health and longevity.

scholarly journals Granule regulation by phase separation during Drosophila oogenesis

2020 ◽  
Vol 4 (3) ◽  
pp. 355-364
Author(s):  
M. Sankaranarayanan ◽  
Timothy T. Weil
Keyword(s):  
Phase Separation ◽  
Translational Control ◽  
Protein Complexes ◽  
Physiological Role ◽  
Drosophila Oogenesis ◽  
And Function ◽  
Rna Protein Complexes ◽  
Liquid Liquid Phase Separation

Drosophila eggs are highly polarised cells that use RNA–protein complexes to regulate storage and translational control of maternal RNAs. Ribonucleoprotein granules are a class of biological condensates that form predominantly by intracellular phase separation. Despite extensive in vitro studies testing the physical principles regulating condensates, how phase separation translates to biological function remains largely unanswered. In this perspective, we discuss granules in Drosophila oogenesis as a model system for investigating the physiological role of phase separation. We review key maternal granules and their properties while highlighting ribonucleoprotein phase separation behaviours observed during development. Finally, we discuss how concepts and models from liquid–liquid phase separation could be used to test mechanisms underlying granule assembly, regulation and function in Drosophila oogenesis.

scholarly journals Phase separation of Axin organizes the β-catenin destruction complex

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Junxiu Nong ◽  
Kexin Kang ◽  
Qiaoni Shi ◽  
Xuechen Zhu ◽  
Qinghua Tao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Glycogen Synthase ◽  
Adenomatous Polyposis ◽  
Compelling Evidence ◽  
Glycogen Synthase Kinase 3Β ◽  
Polyposis Coli ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region ◽  
Liquid Liquid Phase Separation

In Wnt/β-catenin signaling, the β-catenin protein level is deliberately controlled by the assembly of the multiprotein β-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid–liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3β, CK1α, and β-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates β-catenin phosphorylation by GSK3β and is critical for the regulation of β-catenin protein stability and thus Wnt/β-catenin signaling.

scholarly journals TDP-43 α-helical structure tunes liquid–liquid phase separation and function

2020 ◽  
Vol 117 (11) ◽  
pp. 5883-5894 ◽  
Author(s):  
Alexander E. Conicella ◽  
Gregory L. Dignon ◽  
Gül H. Zerze ◽  
Hermann Broder Schmidt ◽  
Alexandra M. D’Ordine ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Changes ◽  
Rna Binding ◽  
Helical Structure ◽  
Liquid Phase Separation ◽  
Control Assembly ◽  
And Function ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

scholarly journals 2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 106 ◽  
Author(s):  
Piero Monaco ◽  
Virginie Marcel ◽  
Jean-Jacques Diaz ◽  
Frédéric Catez
Keyword(s):  
Ribosomal Rna ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
In Vitro Translation ◽  
Ribosome Structure ◽  
Quantitative Mapping ◽  
And Function ◽  
The Impact ◽  
First Time

Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.

scholarly journals Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient β-catenin degradation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tie-Mei Li ◽  
Jing Ren ◽  
Dylan Husmann ◽  
John P. Coan ◽  
Or Gozani ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Adenomatous Polyposis Coli ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Intrinsically Disordered ◽  
High Efficient ◽  
Condensate Formation ◽  
Core Components ◽  
Liquid Liquid Phase Separation

Abstract The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scaffolds GSK3β and CK1 to earmark β-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of β-catenin and oncogenesis. However, the molecular mechanism by which APC promotes β-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple β-catenin and Axin interacting sites, undergoes liquid–liquid phase separation (LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and β-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the β-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of β-catenin.

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