Quadruplex folding promotes the condensation of linker histones and DNAs via liquid-liquid phase separation

Mimura, Masahiro; Tomita, Shunsuke; Shinkai, Yoichi; Hosokai, Takuya; Kumeta, Hiroyuki; Saio, Tomohide; Shiraki, Kentaro; Kurita, Ryoji

doi:10.26434/chemrxiv.11822076

Cited by 5 publications

(11 citation statements)

References 41 publications

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“…S2a for representative peptides: K7Y3 with aromatic side chains, K7S3 with hydrophilic side chains, and K7L3 with aliphatic side chains). Considering previous reports, 6,21,22,30,31 this behaviour confirmed that the observed condensates were not solid aggregates but liquid droplets with highly fluidic properties. To further investigate the liquid properties of the droplets, we carried out fluorescence recovery after photobleaching (FRAP) experiments, a method commonly used to measure the mobility of molecules inside droplets.…”

Section: Design Of a Model Llps Systemsupporting

confidence: 87%

“…This is one of the mechanisms thought to underlie various membrane-less organelles and biomolecular condensates in eukaryotic cells [17][18][19] and is often the model of choice for protocell studies. 3,20 We have also recently used this LLPS mechanism to understand the role of the structural features of DNAs in LLPS, 21,22 to elucidate the compartmentalization of enzymes into droplets and its effect on sequential enzyme reactions, 23 and to facilitate the preparation of high-concentration protein solutions for use in protein formulations. 24,25 Here, as scaffolds for complex coacervation, we selected cationic peptides modified with various non-ionic amino acids and DNA of different sequences.…”

Section: Introductionmentioning

confidence: 99%

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Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

Akahoshi

Sugai

Mimura

et al. 2022

Preprint

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Uncovering the sequence-encoded molecular grammar that governs the liquid–liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of ‘phase-separation propensity’ that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (Ccrit) were determined for each peptide. A correlation analysis between these values and known amino-acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend, and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of nonionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.

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Section: Design Of a Model Llps Systemsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

Akahoshi

Sugai

Mimura

et al. 2022

Preprint

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“…For example, π-π interactions also play a major role in the formation of various biomolecular condensates (Chong et al, 2018;Vernon et al, 2018). It is therefore plausible that π-π interactions between the aromatic amino acids of H1 (3 Tyr and 2 Phe in 194 aa; for the sequence of H1, see Supplementary Figure 1) and the nucleobases and/or those between nucleobases promoted LLPS, as has been previously suggested (Mimura et al, 2021). Cation-π interactions may also be involved (Chong et al, 2018;Qamar et al, 2018).…”

Section: Interactions Between Each Dna Component and H1 In The Promotion Of Llpsmentioning

confidence: 62%

“…Histone H1 (H1) from bovine thymus was obtained from Signal Chem (Richmond, BC, Canada). Prior to experiments, the buffer of the supplied H1 solution (1.0 mg/mL) was replaced with the TE buffer according to the previously reported procedure (Mimura et al, 2021). After the replacement, the concentration of H1 was determined from the absorbance at 280 nm using a NanoDrop One C spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, United States) and the molar extinction coefficient at 280 nm (4470 M −1 cm −1 ) calculated by the ProtParam tool.…”

Section: Methodsmentioning

confidence: 99%

“…The role of each constituent components of DNA in LLPS was investigated through the interactions of ssDNA-based oligomers with cationic polypeptide chains. For this purpose, we used a model system in which liquid droplets are formed by mixing linker histone H1 (H1) and ssDNAs (Figure 1A; Shakya and King, 2018b;Turner et al, 2018;Mimura et al, 2021). H1 has a long and a short cationic lysine-rich intrinsically disordered region (IDR) at its Cand N-terminus, respectively (Figure 1B and Supplementary Figure 1).…”

Section: Experimental Designmentioning

confidence: 99%

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Uncharged Components of Single-Stranded DNA Modulate Liquid–Liquid Phase Separation With Cationic Linker Histone H1

Mimura

Tomita

Sugai

et al. 2021

Front. Cell Dev. Biol.

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Liquid–liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

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Phase-Separation Propensity of Non-ionic Amino Acids in Peptide-Based Complex Coacervation Systems

et al. 2023

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Uncovering the sequence-encoded molecular grammar that governs the liquid−liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of "phase-separation propensity" that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (C crit ) were determined for each peptide. A correlation analysis between these values and known amino acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of non-ionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.

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Quadruplex folding promotes the condensation of linker histones and DNAs via liquid-liquid phase separation

Cited by 5 publications

References 41 publications

Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

Uncharged Components of Single-Stranded DNA Modulate Liquid–Liquid Phase Separation With Cationic Linker Histone H1

Phase-Separation Propensity of Non-ionic Amino Acids in Peptide-Based Complex Coacervation Systems

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