scholarly journals Multivalent electrostatic pi–cation interaction between synaptophysin and synapsin is responsible for the coacervation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Goeun Kim ◽  
Sang-Eun Lee ◽  
Seonyoung Jeong ◽  
Jeongkun Lee ◽  
Daehun Park ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Underlying Mechanism ◽  
Living Cells ◽  
Synaptic Activity ◽  
Physical Interaction ◽  
Liquid Droplets ◽  
High Concentration ◽  
Intrinsically Disordered ◽  
Separating Property

AbstractWe recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid–liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi–cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph’s negative charge, the results indicate that pi–cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi–cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

scholarly journals SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

2015 ◽  
Vol 210 (3) ◽  
pp. 451-470 ◽  
Author(s):  
Miriam Lee ◽  
Young-Joon Ko ◽  
Yeojin Moon ◽  
Minsoo Han ◽  
Hyung-Wook Kim ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Underlying Mechanism ◽  
In Vitro Assay ◽  
Physical Interaction ◽  
Physiological Concentration ◽  
Vitro Assay ◽  
Lack Of Fusion ◽  
Fold Excess

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

scholarly journals CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism

2021 ◽  
Vol 11 ◽  
Author(s):  
Tuersunayi Abudureheman ◽  
Jing Xia ◽  
Ming-Hao Li ◽  
Hang Zhou ◽  
Wei-Wei Zheng ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Apoptosis ◽  
Acute Lymphocytic Leukemia ◽  
Cellular Metabolism ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
High Concentration ◽  
P53 Expression

B-cell acute lymphocytic leukemia (B-ALL) is a malignant blood cancer that develops in children and adults and leads to high mortality. THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, shows anti-tumor effects in various cancers by inhibiting cell proliferation and inducing apoptosis. However, whether THZ1 has an inhibitory effect on B-ALL cells and the underlying mechanism remains obscure. In this study, we showed that THZ1 arrested the cell cycle of B-ALL cells in vitro in a low concentration, while inducing the apoptosis of B-ALL cells in vitro in a high concentration by activating the apoptotic pathways. In addition, RNA-SEQ results revealed that THZ1 disrupted the cellular metabolic pathways of B-ALL cells. Moreover, THZ1 suppressed the cellular metabolism and blocked the production of cellular metabolic intermediates in B-ALL cells. Mechanistically, THZ1 inhibited the cellular metabolism of B-ALL by downregulating the expression of c-MYC-mediated metabolic enzymes. However, THZ1 treatment enhanced cell apoptosis in over-expressed c-MYC B-ALL cells, which was involved in the upregulation of p53 expression. Collectively, our data demonstrated that CDK7 inhibitor THZ1 induced the apoptosis of B-ALL cells by perturbing c-MYC-mediated cellular metabolism, thereby providing a novel treatment option for B-ALL.

scholarly journals Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

2021 ◽  
Author(s):  
Aishwarya Agarwal ◽  
Lisha Arora ◽  
Sandeep K. Rai ◽  
Anamika Avni ◽  
Samrat Mukhopadhyay
Keyword(s):  
Electrostatic Interactions ◽  
Physiological Role ◽  
Terminal Segment ◽  
Liquid Droplets ◽  
Cellular Functions ◽  
Time Resolved ◽  
Domain Specific ◽  
Intrinsically Disordered ◽  
Synergistic Interactions ◽  
Heterotypic Interactions

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) of proteins and nucleic acids are thought to govern critical cellular functions. These multicomponent assemblies provide dynamic hubs for competitive homotypic and heterotypic interactions. Here, we demonstrate that the complex coacervation between the prion protein (PrP) and α-synuclein (α-Syn) within a narrow stoichiometry regime results in the formation of highly dynamic liquid droplets. Domain-specific electrostatic interactions between the positively charged intrinsically disordered N-terminal segment of PrP and the negatively charged C-terminal domain of α-Syn drive the formation of these highly tunable, reversible, thermo-responsive condensates. Picosecond time-resolved measurements revealed the existence of relatively ordered electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making on a much slower timescale giving rise to the liquid-like behavior on the second timescale and mesoscopic length-scale. The addition of RNA to these preformed coacervates yields multiphasic, anisotropic, vesicle-like, hollow condensates. LLPS promotes liquid-to-solid maturation of α-Syn-PrP condensates resulting in the rapid conversion into heterotypic amyloids. Our results suggest that synergistic interactions between PrP and α-Syn in liquid condensates can offer mechanistic underpinnings of their physiological role and overlapping neuropathological features.

scholarly journals Oligomerization of the Human Adenosine A2A Receptor is Driven by the Intrinsically Disordered C-terminus

2020 ◽  
Author(s):  
Khanh D. Q. Nguyen ◽  
Michael Vigers ◽  
Eric Sefah ◽  
Susanna Seppälä ◽  
Jennifer P. Hoover ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Intrinsically Disordered ◽  
Interaction Sites ◽  
C Terminus ◽  
A2a Receptor ◽  
Multiple Interaction ◽  
Adenosine A2a

ABSTRACTG protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of GPCR oligomerization remain relatively unexplored. In this study, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives the homo-oligomerization of the receptor. The formation of A2AR oligomers declines progressively and systematically with the shortening of the C-terminus. Multiple interaction sites and types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions along the C-terminus, forming a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important guidance for structure-function studies of A2AR and other GPCRs.

scholarly journals Conserved tau microtubule-binding repeat histidines confer pH-dependent tau-microtubule association

2018 ◽  
Author(s):  
Rabab A. Charafeddine ◽  
Wilian A. Cortopassi ◽  
Parnian Lak ◽  
Matthew P. Jacobson ◽  
Diane L. Barber ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Electrostatic Interactions ◽  
Cell Model ◽  
Interaction Strength ◽  
Microtubule Binding ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Decreasing Ph ◽  
Positively Charged

ABSTRACTTau, a member of the MAP2/tau family of microtubule-associated proteins, functions to stabilize and organize axonal microtubules in healthy neurons. In contrast, tau dissociates from microtubules and forms neurotoxic extracellular aggregates in neurodegenerative tauopathies. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. We use molecular dynamics, microtubule-binding experiments and live cell microscopy to show that highly conserved histidine residues near the C terminus of each MT-binding repeat are pH sensors that can modulate tau-MT interaction strength within the physiological intracellular pH range. At lower pH, these histidines are positively charged and form cation-π interactions with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH, tau deprotonation decreases microtubule-binding both in vitro and in cells. However, electrostatic and hydrophobic characteristics of histidine are required for tau-MT-binding as substitution with constitutively positively charged, non-aromatic lysine or uncharged alanine greatly reduces or abolishes tau-MT binding. Consistent with these findings, tau-MT binding is reduced in a cancer cell model with increased intracellular pH but is rapidly rescued by decreasing pH to normal levels. Thus, these data add a new dimension to the intracellular regulation of tau activity and could be relevant in normal and pathological conditions.

scholarly journals Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

2017 ◽  
Author(s):  
Shasha Chong ◽  
Claire Dugast-Darzacq ◽  
Zhe Liu ◽  
Peng Dong ◽  
Gina M. Dailey ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Low Complexity ◽  
Live Cell ◽  
Single Molecule Imaging ◽  
High Concentration ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Dynamic Display

AbstractMany eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occur between TFs and the Pol II machinery, which underpins a central mechanism for transactivation and plays a key role in gene expression and disease.

scholarly journals The mechanism underlying the organization of Borna disease virus inclusion bodies is unique among mononegaviruses

2021 ◽  
Author(s):  
Yuya Hirai ◽  
Keizo Tomonaga ◽  
Masayuki Horie
Keyword(s):  
Disease Virus ◽  
Inclusion Bodies ◽  
Liquid Droplets ◽  
Borna Disease Virus ◽  
Intrinsically Disordered ◽  
Excess Amount ◽  
Nucleoprotein N ◽  
Borna Disease ◽  
Liquid Liquid Phase Separation

Inclusion bodies (IBs) are characteristic biomolecular condensates organized by mononegaviruses. Here, we characterize the IBs of Borna disease virus 1 (BoDV-1), a unique mononegavirus that forms IBs in the nucleus, in terms of liquid-liquid phase separation (LLPS). The BoDV-1 phosphoprotein (P) alone induces LLPS and the nucleoprotein (N) is incorporated into the P droplet in vitro. In contrast, co-expression of N and P is required for the formation of IB-like structure in cells. Furthermore, while BoDV-1 P binds to RNA, an excess amount of RNA dissolves the liquid droplets formed by N and P. Notably, the N-terminal intrinsically disordered region of BoDV-1 P is essential to drive LLPS and bind to RNA, suggesting that both abilities could compete with one another. These features are unique among mononegaviruses, and thus this study will contribute to a deeper understanding of LLPS-driven organization and RNA-mediated regulation of biomolecular condensates.

scholarly journals Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

2021 ◽  
Vol 17 (3) ◽  
pp. e1008672
Author(s):  
Wen-Ting Chu ◽  
Jin Wang
Keyword(s):  
Phase Separation ◽  
High Temperatures ◽  
Electrostatic Interactions ◽  
Sequence Length ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
Conventional Behavior ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2019 ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Cooperative Assembly

SummaryCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions (DR1-3) that flank positively charged α-helical regions (HR1 and HR2). Here we show that the three DR domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between DR and HR regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1 (CAV1), and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and CAV1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

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