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Acetylation of DDX3X-IDR1 impairs its droplet formation by LLPS in vitro a, Effect of acetylation on LLPS of DDX3X-IDR1. Left, turbidity (OD600) of DDX3X-IDR1 acetylated by CBP (acetylation) and the mock reaction control (mock reaction) in 20 mM Tris, pH 7.5, 125 mM NaCl, 2 mM DTT and 10% PEG was measured at room temperature (mean ± s.d.; n = 3 independent experiments). For the mock reaction control, the IDR1, CBP and acetyl-CoA were mixed directly before the turbidity measurement. Right, phase separation of 100 μM IDR1 solutions (top). Liquid droplets visualized by DIC microscopy (bottom; scale bar, 10 μm). b, Recovery of LLPS by HDAC6-mediated deacetylation. Turbidity (OD600) of DDX3X-IDR1 was measured after the reaction with CBP or CBP + HDAC6 (mean ± s.d.; n = 3 independent experiments). The HDAC6 reaction was carried out at two concentrations (molar ratio between CBP and HDAC6 = 1:0.5 (blue), 1:1 (green)). c, Left, lysine to glutamine (acetyllysine mimic) substitution impairs LLPS of DDX3X-IDR1. Turbidity (OD600) of WT, K118Q, K118R and allQ mutants of DDX3X-IDR1 in 20 mM Tris, pH 7.5, 200 mM NaCl, 2 mM DTT and 10% PEG was measured (mean ± s.d.; n = 3 independent experiments). Right, phase separation of 100 μM IDR1 solutions (top) and liquid droplets visualized by DIC microscopy (bottom; scale bar, 10 μm). d, Percent of turbidity (OD600) of DDX3X-IDR1 mutants from Supplementary Fig. 12b, normalized to WT (mean ± s.d.; n = 3 independent experiments). P values were determined by Student’s two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. e, Acetylation of the DDX3X IDR regulates its ability to precipitate with biotinylated isoxazole (b-isox) in vitro. Left, lysates from 293T cells expressing GFP (control) or CBP were subjected to b-isox-mediated precipitation and analyzed by immunoblotting. The presence of endogenous DDX3X-K118Ac was assessed in the precipitate fraction (b-isox ppt) and in the lysate fraction (input) and compared to the level of total precipitated DDX3X. TIAR was used as a control protein harboring an IDR, whereas ubiquitin and α-tubulin were used as control proteins lacking an IDR. Proteins were detected with specific antibodies, as indicated. Representative results are shown (n = 3 biologically independent samples). Uncropped gel images can be found in Supplementary Fig. 27g. Right, schematic illustrating the b-isox-mediated precipitation of unacetylated DDX3X IDRs with acetylated IDRs remaining in suspension.
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Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LL...
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Citations
... So, Smad2/3/4 complex must stay on phosphorylation status when the complex translocates into the nucleus and regulates target gene expression. Phosphorylation and methylation are important for protein undergoing LLPS [43][44][45][46]. In our research, we used E. coli to express Smad2/3/4 recombinant protein. ...
Background
Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved.
Methods
We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo.
Results
We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis.
Conclusion
This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development.
... In biochemical experiments, such IDRs show the ability to phase separate into liquid-like assemblies [35][36][37][38][39][40][41][42][43][44][45]. The accessibility or activities of IDRs can be tightly regulated by posttranslational modifications, allowing rapid physiological and spatial control over granule assembly and disassembly [29,35,[46][47][48][49][50][51][52]. An important observation is that most IDRs also can transition from liquid-like state into solid, beta-sheet rich, amyloid-fibrils in vitro, particularly at high concentrations achieved in the liquid phase [22,41,[53][54][55]. ...
Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.
... 21,22 Due to the well-recognized role of electrostatic interactions in modulating protein phase-separation, the significance of 'charge-altering' PTMs is under intense investigation and is beginning to come into a sharper focus. These include but are not limited to, serine phosphorylation, 23,24 arginine-methylation, 25 and lysine acetylation, 26 which modify multivalent electrostatic interactions by altering columbic charges or their distributions. However, more than 300 types of PTMs have been identified, and many do not alter the electrostatic charge significantly. ...
Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)—to modulate the amino acid physicochemistry instead of altering protein sequences—to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation—in synergy with the sequence of the lipidation site—significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of “sequence context” in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.
... As an important member of the DDX family, the human DDX3X is a multifunctional helicase exhibiting broader roles in RNA metabolism, cellular stress responses, and innate immunity [15,16]. DDX3X's involvement in LLPS is particularly significant, as it influences the assembly and dynamics of stress granules, which are critical under conditions of cellular stress [17,18]. Its yeast homolog, DED1, is essential for mRNA splicing and translation initiation [19][20][21]. ...
The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Bioinformatic analysis of cancer genomic datasets uncovered a significant enrichment of DDX3X mutations in RG/RGG motifs across multiple cancer types, particularly in the N-terminal region of the protein. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.
... To further explore the mechanisms of V1 potentiation, we evaluated four additional mammalian IDRs, derived from DDX4, DDX3X, FUS, and HNRNPA1, respectively; these IDRs are all well-defined, and their sequences are unrelated ( Figure S7A). 24, 25 We fused a single copy of these IDRs to V1, and compared their effects with that of 1Â CYCT1 HRD. The IDRs from DDX4 and DDX3X behaved similarly to the CYCT1 HRD. ...
RNA base editors should ideally be free of immunogenicity, compact, efficient, and specific, which has not been achieved for C > U editing. Here we first describe a compact C > U editor entirely of human origin, created by fusing the human C > U editing enzyme RESCUE-S to Cas inspired RNA targeting system (CIRTS), a tiny, human-originated programmable RNA-binding domain. This editor, CIRTS-RESCUEv1 (V1), was inefficient. Remarkably, a short histidine-rich domain (HRD), which is derived from the internal disordered region (IDR) in the human CYCT1, a protein capable of liquid-liquid phase separation (LLPS), enhanced V1 editing at on-targets as well as off-targets, the latter effect being minor. The V1-HRD fusion protein formed puncta characteristic of LLPS, and various other IDRs (but not an LLPS-impaired mutant) could replace HRD to effectively induce puncta and potentiate V1, suggesting that the diverse domains acted via a common, LLPS-based mechanism. Importantly, the HRD fusion strategy was applicable to various other types of C > U RNA editors. Our study expands the RNA editing toolbox and showcases a general method for stimulating C > U RNA base editors.
... Oligolysine consists solely of lysine (K) residue, which are positively charged under physiological conditions and it role in the field of biological phase separation has been well recognized. 17,18 ATP is wellknown for its role as an energy currency. Its high abundance in the cytosol, along with its biphasic regulatory effects on protein stability, solubility, and aggregation propensity, renders it a promising species. ...
Liquid-Liquid Phase Separation (LLPS) plays a pivotal role in the organization and functionality of living cells. It is imperative to understand the underlying driving forces behind LLPS and to control...
... Acetylation and methylation are two very common modifications of histone proteins that determine chromatin accessibility and thus the rate of transcription (113). The subcompartmentalization of differentially active chromatin is driven by the phase separation, suggesting that these PTMs are indeed critical features that define the dynamic nuclear architecture and influence gene expression (114,115). The above data show that PIP2-associated IDRs are sites of intense PTM regulation, suggesting their importance in regulating processes that depend on protein condensation capacity. ...
The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation and ubiquitination sites. Our findings reveal that RDPA protein BRD4 associates with PIP2 in an RNA-dependent manner via electrostatic interactions, and that elevated PIP2 levels increase the number of BRD4 protein nuclear foci. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to phase separate. This suggests pivotal role of PIP2 for the establishment of a functional nuclear architecture competent for gene expression.
... This dual-domain configuration is an exclusive trait amongst HDACs, endowing HDAC6 with an enhanced and diversified substrate specificity. For instance, DEAD-box helicase 3 X-linked (DDX3X) has been identified as a substrate for both CD1 and CD2 domains [11], while α-tubulin, cortactin, and heat shock protein 90 (HSP 90) are known as specific substrates for the CD2 domain [4,9,10,12,13]. Beyond its deacetylase domains, HDAC6 incorporates a cytoplasmic anchoring domain, predominantly localizing the protein within the cytoplasm, as well as a zinc finger motif (ZNF) for ubiquitin recognition which is involved in protein degradation and autophagy processes [14][15][16]. ...
Histone deacetylase 6 (HDAC6) plays a crucial role in the acetylation of non-histone proteins and is notably implicated in angiogenesis, though its underlying mechanisms were previously not fully understood. This study conducted transcriptomic and proteomic analyses on vascular endothelial cells with HDAC6 knockdown, identifying endoglin (ENG) as a key downstream protein regulated by HDAC6. This protein is vital for maintaining vascular integrity and plays a complex role in angiogenesis, particularly in its interaction with bone morphogenetic protein 9 (BMP9). In experiments using human umbilical vein endothelial cells (HUVECs), the pro-angiogenic effects of BMP9 were observed, which diminished following the knockdown of HDAC6 and ENG. Western blot analysis revealed that BMP9 treatment increased SMAD1/5/9 phosphorylation, a process hindered by HDAC6 knockdown, correlating with reduced ENG expression. Mechanistically, our study indicates that HDAC6 modulates ENG transcription by influencing promoter activity, leading to increased acetylation of transcription factor SP1 and consequently altering its transcriptional activity. Additionally, the study delves into the structural role of HDAC6, particularly its CD2 domain, in regulating SP1 acetylation and subsequently ENG expression. In conclusion, the present study underscores the critical function of HDAC6 in modulating SP1 acetylation and ENG expression, thereby significantly affecting BMP9-mediated angiogenesis. This finding highlights the potential of HDAC6 as a therapeutic target in angiogenesis-related processes.
... Acetylation on multiple lysine residues of the N-terminal IDR of DDX3X (IDR1) impairs droplet formation. And the enhancement of LLPS propensity by deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation [86]. In terms of gene expression, p300 histone acetylation antagonizes chromatin phase separation and reduces the formation of droplets in the nucleus [87]. ...
The phenomenon of phase separation is quite common in cells, and it is involved in multiple processes of life activities. However, the current research on the correlation between protein modifications and phase separation and the interference with the tendency of phase separation has some limitations. Here we focus on several post-translational modifications of proteins, including protein phosphorylation modification at multiple sites, methylation modification, acetylation modification, ubiquitination modification, SUMOylation modification, etc., which regulate the formation of phase separation and the stability of phase separation structure through multivalent interactions. This regulatory role is closely related to the development of neurodegenerative diseases, tumors, viral infections, and other diseases, and also plays essential functions in environmental stress, DNA damage repair, transcriptional regulation, signal transduction, and cell homeostasis of living organisms, which provides an idea to explore the interaction between novel protein post-translational modifications and phase separation.
... There are several types of PTMs that can occur within IDRs, including phosphorylation, acetylation, methylation, ubiquitination, and many others. These modifications can introduce changes to specific amino acid residues within the IDRs, altering their physicochemical properties and affecting protein-protein interactions [20,21]. The phenomenon of multivalence arises from the presence of diverse repetitive interaction sites within a polypeptide or nucleic acid molecule, which can be achieved through folded binding modules or IDRs. ...
DNA double-strand break (DSB) is the most dangerous type of DNA damage, which may lead to cell death or oncogenic mutations. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two typical DSB repair mechanisms. Recently, many studies have revealed that liquid–liquid phase separation (LLPS) plays a pivotal role in DSB repair and response. Through LLPS, the crucial biomolecules are quickly recruited to damaged sites with a high concentration to ensure DNA repair is conducted quickly and efficiently, which facilitates DSB repair factors activating downstream proteins or transmitting signals. In addition, the dysregulation of the DSB repair factor’s phase separation has been reported to promote the development of a variety of diseases. This review not only provides a comprehensive overview of the emerging roles of LLPS in the repair of DSB but also sheds light on the regulatory patterns of phase separation in relation to the DNA damage response (DDR).
