scholarly journals Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Minglei Shi ◽  
Kaiqiang You ◽  
Taoyu Chen ◽  
Chao Hou ◽  
Zhengyu Liang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Quantitative Proteomics ◽  
Quantitative Measurement ◽  
Global Analysis ◽  
Active Chromatin ◽  
Future Studies ◽  
Physiological Conditions ◽  
Associated Proteins ◽  
Order Structures ◽  
Liquid Liquid Phase Separation

Background Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. Results Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. Conclusions Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

scholarly journals Quantifying liquid-liquid phase separation property of chromatin under physiological conditions using Hi-MS and Hi-C

2020 ◽  
Author(s):  
Minglei Shi ◽  
Kaiqiang You ◽  
Chao Hou ◽  
Taoyu Chen ◽  
Zhengyu Liang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Quantitative Measurement ◽  
Biological Process ◽  
Global Analysis ◽  
Chromatin Organization ◽  
Liquid Phase Separation ◽  
Protein Functions ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation

AbstractBackgroundLiquid–liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent.ResultsHere, by combing chromatin associated protein pull-down, quantitative proteomics and 1,6-hexanediol treatment, we developed Hi-MS and defined anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantitative measurement of LLPS property of chromatin-associated proteins in their endogenous state and physiological abundance. The AICAP values were verified by previously reported experiments and were reproducible across different MS platforms. Moreover, the AICAP values were highly correlate with protein functions. Proteins act in active/regulatory biological process often exhibit low AICAP values, while proteins act in structural and repressed biological process often exhibit high AICAP values. We further revealed that chromatin organization changes more in compartment A than B, and the changes in chromatin organization at various levels, including compartments, TADs and loops are highly correlated to the LLPS properties of their neighbor nuclear condensates.ConclusionsOur work provided the first global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure, and demonstrate that the active/regulatory chromatin components, both protein (trans) and DNA (cis), exhibit more hydrophobicity-dependent LLPS properties than the repressed/structural chromatin components.

scholarly journals Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

2021 ◽  
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation ◽  
In Cells ◽  
Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

scholarly journals Liquid-liquid phase separation and fibrillization of tau are independent processes with overlapping conditions

2019 ◽  
Author(s):  
Yanxian Lin ◽  
Yann Fichou ◽  
Zhikai Zeng ◽  
Nicole Y. Hu ◽  
Songi Han
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Amyloid Aggregation ◽  
Physiological Conditions ◽  
Three Stages ◽  
Tau Aggregation ◽  
Kinetics Of ◽  
Liquid Liquid Phase Separation

AbstractAmyloid aggregation of the microtubule binding protein tau is a hallmark of Alzheimer’s disease and many other neurodegenerative diseases. Recently, tau has been found to undergo liquid-liquid phase separation (LLPS) near physiological conditions. Although LLPS and aggregation have been shown to simultaneously occur under certain common conditions, it remains to be seen whether tau LLPS promotes aggregation, or if they are two independent processes. In this study, we address this question by combining multiple biochemical and biophysical assays in vitro. We investigated the impacts of LLPS on tau aggregation at three stages: conformation of tau, kinetics of aggregation and fibril quantity. We showed that none of these properties are influenced directly by LLPS, while amyloid aggregation propensity of tau can be altered without affecting its LLPS behavior. LLPS and amyloid aggregation of tau occur under overlapping conditions of enhanced intermolecular interactions and localization, but are two independent processes.

scholarly journals ATP regulates TDP-43 pathogenesis by specifically binding to an inhibitory component of a delicate network controlling LLPS

2020 ◽  
Author(s):  
Dang Mei ◽  
Liangzhong Lim ◽  
Jian Kang ◽  
Jianxing Song
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Neuron Death ◽  
Physiological Conditions ◽  
Intrinsically Disordered ◽  
Physicochemical Features ◽  
First Time ◽  
Inhibitory Interactions ◽  
Liquid Liquid Phase Separation

ABSTRACTVery recently, liquid-liquid phase separation (LLPS) of cytoplasmic TDP-43 independent of forming stress granule (SG) has been decoded to initiate the neuron death for ALS. Mysteriously neurons maintain ATP concentrations of ∼3 mM, but whether ATP modulates TDP-43 LLPS remains completely unknown. Here we characterized the effects of ATP on LLPS of TDP-43 PLD and its six mutants by DIC and NMR. For the first time, the results revealed: 1) ATP does induce and subsequently dissolve LLPS of TDP-43 PLD. 2) ATP achieves the modulation all by specifically binding Arg mostly saturated at 1:100. 3) LLPS of TDP-43 PLD and its exaggeration into aggregation are controlled by a delicate network composed of both attractive and inhibitory interactions, thus rationalizing the susceptibility of TDP-43 PLD to various ALS-causing mutations. Our studies together establish that ATP specifically binds Arg in intrinsically-disordered domains even not RGG-/R-rich, implying that ATP might be a universal regulator for most, if not all, R-containing intrinsically-disordered domains by altering their physicochemical features, conformations, dynamics, LLPS and assembly. Under physiological conditions, TDP-43 even in neuronal cytoplasm is highly bound with ATP and thus inhibited for its toxic LLPS, highlighting a central role of ATP in TDP-43 pathogenesis and aging.

scholarly journals G-quadruplex structures trigger RNA phase separation

2019 ◽  
Author(s):  
Yueying Zhang ◽  
Minglei Yang ◽  
Susan Duncan ◽  
Xiaofei Yang ◽  
Mahmoud A S Abdelhamid ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Molecular Basis ◽  
Short Root ◽  
Physiological Conditions ◽  
Cellular Processes ◽  
G Quadruplex ◽  
Liquid Liquid Phase Separation ◽  
In Cells

Abstract Liquid–liquid phase separation plays an important role in a variety of cellular processes, including the formation of membrane-less organelles, the cytoskeleton, signalling complexes, and many other biological supramolecular assemblies. Studies on the molecular basis of phase separation in cells have focused on protein-driven phase separation. In contrast, there is limited understanding on how RNA specifically contributes to phase separation. Here, we described a phase-separation-like phenomenon that SHORT ROOT (SHR) RNA undergoes in cells. We found that an RNA G-quadruplex (GQ) forms in SHR mRNA and is capable of triggering RNA phase separation under physiological conditions, suggesting that GQs might be responsible for the formation of the SHR phase-separation-like phenomenon in vivo. We also found the extent of GQ-triggered-phase-separation increases on exposure to conditions which promote GQ. Furthermore, GQs with more G-quartets and longer loops are more likely to form phase separation. Our studies provide the first evidence that RNA can adopt structural motifs to trigger and/or maintain the specificity of RNA-driven phase separation.

scholarly journals Therapeutics—how to treat phase separation-associated diseases

2020 ◽  
Vol 4 (3) ◽  
pp. 331-342 ◽  
Author(s):  
Richard John Wheeler
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Conceptual Framework ◽  
Protein Function ◽  
Enzyme Inhibitors ◽  
Liquid Phase Separation ◽  
Post Translational Modification ◽  
New Paradigm ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation has drawn attention as many neurodegeneration or cancer-associated proteins are able to form liquid membraneless compartments (condensates) by liquid–liquid phase separation. Furthermore, there is rapidly growing evidence that disease-associated mutation or post-translational modification of these proteins causes aberrant location, composition or physical properties of the condensates. It is ambiguous whether aberrant condensates are always causative in disease mechanisms, however they are likely promising potential targets for therapeutics. The conceptual framework of liquid–liquid phase separation provides opportunities for novel therapeutic approaches. This review summarises how the extensive recent advances in understanding control of nucleation, growth and composition of condensates by protein post-translational modification has revealed many possibilities for intervention by conventional small molecule enzyme inhibitors. This includes the first proof-of-concept examples. However, understanding membraneless organelle formation as a physical chemistry process also highlights possible physicochemical mechanisms of intervention. There is huge demand for innovation in drug development, especially for challenging diseases of old age including neurodegeneration and cancer. The conceptual framework of liquid–liquid phase separation provides a new paradigm for thinking about modulating protein function and is very different from enzyme lock-and-key or structured binding site concepts and presents new opportunities for innovation.

scholarly journals PhaSepDB: a database of liquid–liquid phase separation related proteins

2019 ◽  
Vol 48 (D1) ◽  
pp. D354-D359 ◽  
Author(s):  
Kaiqiang You ◽  
Qi Huang ◽  
Chunyu Yu ◽  
Boyan Shen ◽  
Cristoffer Sevilla ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Online Database ◽  
Sequence Features ◽  
The Past ◽  
Associated Proteins ◽  
Related Proteins ◽  
Convenient Interface ◽  
Liquid Liquid Phase Separation

Abstract It's widely appreciated that liquid–liquid phase separation (LLPS) underlies the formation of membraneless organelles, which function to concentrate proteins and nucleic acids. In the past few decades, major efforts have been devoted to identify the phase separation associated proteins and elucidate their functions. To better utilize the knowledge dispersed in published literature, we developed PhaSepDB (http://db.phasep.pro/), a manually curated database of phase separation associated proteins. Currently, PhaSepDB includes 2914 non-redundant proteins localized in different organelles curated from published literature and database. PhaSepDB provides protein summary, publication reference and sequence features of phase separation associated proteins. The sequence features which reflect the LLPS behavior are also available for other human protein candidates. The online database provides a convenient interface for the research community to easily browse, search and download phase separation associated proteins. As a centralized resource, we believe PhaSepDB will facilitate the future study of phase separation.

scholarly journals Liquid-Liquid Phase Separation of Wheat Gliadins - Towards Physiological Conditions

2020 ◽  
Vol 118 (3) ◽  
pp. 485a
Author(s):  
Line Sahli ◽  
Denis Renard ◽  
Véronique Solé-Jamault ◽  
Adeline Boire
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Physiological Conditions ◽  
Liquid Liquid Phase Separation

scholarly journals DrLLPS: a data resource of liquid–liquid phase separation in eukaryotes

2019 ◽  
Vol 48 (D1) ◽  
pp. D288-D295 ◽  
Author(s):  
Wanshan Ning ◽  
Yaping Guo ◽  
Shaofeng Lin ◽  
Bin Mei ◽  
Yu Wu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Model Organisms ◽  
Post Translational Modifications ◽  
Data Resource ◽  
Functional Annotations ◽  
Cancer Mutations ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation

Abstract Here, we presented an integrative database named DrLLPS (http://llps.biocuckoo.cn/) for proteins involved in liquid–liquid phase separation (LLPS), which is a ubiquitous and crucial mechanism for spatiotemporal organization of various biochemical reactions, by creating membraneless organelles (MLOs) in eukaryotic cells. From the literature, we manually collected 150 scaffold proteins that are drivers of LLPS, 987 regulators that contribute in modulating LLPS, and 8148 potential client proteins that might be dispensable for the formation of MLOs, which were then categorized into 40 biomolecular condensates. We searched potential orthologs of these known proteins, and in total DrLLPS contained 437 887 known and potential LLPS-associated proteins in 164 eukaryotes. Furthermore, we carefully annotated LLPS-associated proteins in eight model organisms, by using the knowledge integrated from 110 widely used resources that covered 16 aspects, including protein disordered regions, domain annotations, post-translational modifications (PTMs), genetic variations, cancer mutations, molecular interactions, disease-associated information, drug-target relations, physicochemical property, protein functional annotations, protein expressions/proteomics, protein 3D structures, subcellular localizations, mRNA expressions, DNA & RNA elements, and DNA methylations. We anticipate DrLLPS can serve as a helpful resource for further analysis of LLPS.

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