Formation Of Oligomers Research Articles

TSH Receptor Oligomers Associated With the TSH Receptor Antibody Reactome.

The TSH receptor (TSHR) and its many forms are the primary antigens of Graves' disease as evidenced by the presence of TSHR antibodies of differing biological activity. The TSH holoreceptor undergoes complex posttranslational changes including cleavage of its ectodomain and oligomer formation. We have previously shown that the TSHR exists in both monomeric and dimeric structures in the thyroid cell membrane and have demonstrated, by modeling, that the transmembrane domains (TMD) can form stable dimeric structures. Based on these earlier simulations of the TSHR-TMD structure and our most recent model of the full-length TSHR, we have now built models of full-length TSHR multimers with and without TSH ligand in addition to multimers of the extracellular leucine-rich domain, the site of TSH and autoantibody binding. Starting from these models we ran molecular dynamics simulations of the receptor oligomers solvated with water and counterions; the full-length oligomers also were embedded in a dipalmitoylphosphatidylcholine bilayer. The full-length TSHR dimer and trimer models stayed in the same relative orientation and distance during 2000 ns (or longer) molecular dynamics simulation in keeping with our earlier report of TMD dimerization. Simulations were also performed to model oligomers of the leucine-rich domain alone; we found a trimeric complex to be even more stable than the dimers. These data provide further evidence that different forms of the TSHR add to the complexity of the immune response to this antigen that, in patients with autoimmune thyroid disease, generate an autoantibody reactome with multiple types of autoantibody to the TSHR.

Chaperone BiP controls ER stress sensor Ire1 through interactions with its oligomers.

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

Moment dynamics of oligomer formation in protein amyloid aggregation with secondary nucleation

The abnormal aggregation of proteins into amyloid fibrils, usually implemented by a series of biochemical reactions, is associated with various neurodegenerative disorders. Considering the intrinsic stochasticity in the involving biochemical reactions, a general chemical master equation model for describing the process from oligomer production to fibril formation is established, and then the lower-order statistical moments of different molecule species are captured by the derivative matching closed system, and the long-time accuracy is verified using the Gillespie algorithm. It is revealed that the aggregation of monomers into oligomers is highly dependent on the initial number of misfolded monomers; the formation of oligomers can be effectively inhibited by reducing the misfolding rate, the primary nucleation rate, elongation rate, and secondary nucleation rate; as the conversion rate decreases, the number of oligomers increases over a long time scale. In particular, sensitivity analysis shows that the quantities of oligomers are more sensitive to monomer production and protein misfolding; the secondary nucleation is more important than the primary nucleation in oligomer formation. These findings are helpful for understanding and predicting the dynamic mechanism of amyloid aggregation from the viewpoint of quantitative analysis.

Targeting the hydrophobic region of pyroglutamate-modified amyloid-β by tyrocidine A prevents its nucleation-aggregation process and its "catalytic effect" on the Aβsaggregation.

Pyroglutamate (pE)-modified amyloid-β (Aβ) peptides play a crucial role in the development of Alzheimer's disease. pEAβ3-42 can rapidly form oligomers that gradually elongate hydrophobic segments to form β-sheet-rich amyloid intermediates, ultimately resulting in the formation of mature amyloid fibrils. pEAβ3-42 can also catalyze the aggregation of Aβ species and subsequently accelerate the formation of amyloid senile plaques. Considering the recent clinical success of the pEAβ3-42-targeting antibody donanemab, molecules that strongly bind pEAβ3-42 and prevent its aggregation and catalytic effect on Aβs may also provide potential therapeutic options for Alzheimer's disease. Here, we demonstrate that the natural antibiotic cyclopeptide tyrocidine A (TA) not only strongly inhibits the aggregation of Aβ1-42 as previously reported, but also interacts with the hydrophobic C-terminus and middle domain of pEAβ3-42 to maintain an unordered conformation, effectively impeding the formation of initial oligomers and subsequently halting the aggregation of pEAβ3-42. Furthermore, TA can disrupt the "catalytic effect" of pEAβ3-42 on amyloid aggregates, effectively suppressing Aβaggregation and ultimately preventing the pathological events induced by Aβs.

Identification and characterization of transition metal-binding proteins and metabolites in the phloem sap of Brassica napus

Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography (SEC) coupled online to sector-field ICP-MS. Our data identified known TM binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10-15 kD containing Cu, Fe, S and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

Monomer diffusion tendencies in complex-shaped materials by vapor deposition polymerization

Conformal coatings are crucial for various applications. In this study, we investigate polyurea coatings on complex-shape polytetrafluoroethylene (PTFE) membranes using vapor deposition polymerization. The diffusion mechanism involves monomer adsorption and desorption on PTFE fibers, enhanced by an increased substrate temperature, facilitating monomer diffusion. A system pressure of 100 Pa promoted monomer collisions, forming oligomers with lower desorption rates, aiding polymer thin-film formation. The combination of high substrate temperature for diffusion and gas-phase oligomer formation resulted in uniform polyurea coatings across the PTFE membrane. A non-exhaustion method provided an extensive monomer diffusion, achieving a thorough polyurea deposition throughout the membrane structure.

Cadmium causes cerebral mitochondrial dysfunction through regulating mitochondrial HSF1

Mitochondria, as the powerhouse of the cell, play a vital role in maintaining cellular energy homeostasis and are known to be a primary target of cadmium (Cd) toxicity. The improper targeting of proteins to mitochondria can compromise the normal functions of the mitochondria. However, the precise mechanism by which protein localization contributes to the development of mitochondrial dysfunction induced by Cd is still not fully understood. For this research, Hy-Line white variety chicks (1-day-old) were used and equally distributed into 4 groups: the Control group (fed with a basic diet), the Cd35 group (basic diet with 35 mg/kg CdCl2), the Cd70 group (basic diet with 70 mg/kg CdCl2) and the Cd140 group (basic diet with 140 mg/kg CdCl2), respectively for 90 days. It was found that Cd caused the accumulation of heat shock factor 1 (HSF1) in the mitochondria, and the overexpression of HSF1 in the mitochondria led to mitochondrial dysfunction and neuronal damage. This process is due to the mitochondrial HSF1 (mtHSF1), causing mitochondrial fission through the upregulation of dynamin-related protein 1 (Drp1) content, while inhibiting oligomer formation of single-stranded DNA-binding protein 1 (SSBP1), resulting in the mitochondrial DNA (mtDNA) deletion. The findings unveil an unforeseen role of HSF1 in triggering mitochondrial dysfunction.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among them, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here, we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72), and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive, while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

Guluronic acid disaccharide inhibits reactive oxygen species production and amyloid-β oligomer formation

In general, Cu(II) is the critical factor in catalyzing reactive oxygen species (ROS) production and accelerating amyloid-β (Aβ) oligomer formation in Alzheimer's disease (AD). Natural chelating agents with good biocompatibility and appropriate binding affinity with Cu(II) have emerged as potential candidates for AD therapy. Herein, we tested the capability of guluronic acid disaccharide (Di–GA), a natural chelating agent with the moderate association affinity to Cu(II), in inhibiting ROS production and Aβ oligomer formation. The results showed that Di−GA was capable of chelating with Cu(II) and reducing ROS production, even in solutions containing Fe(II), Zn(II), and Aβ. In addition, Di−GA can also capture Cu(II) from Cu−Aβ complexes and decrease Aβ oligomer formation. The cellular results confirmed that Di−GA prevented SH−SY5Y cells from ROS and Aβ oligomer damage by reducing the injury of ROS and Aβ oligomers on cell membrane and decreasing their intracellular damage on mitochondria. Notably, the slightly higher efficiency of Di–GA in chelating with Cu(I) than Cu(II) can be benefit for its in vivo applications, as Cu(I) is not only the most active but also the special intermediate specie during ROS production process. All of these results proved that Di–GA could be a promising marine drug candidate in reducing copper-related ROS damage and Aβ oligomer toxicity associated with AD.

Disulfide-Driven Charge and Hydrophobicity Rearrangement of Remodeled Membrane Proteins toward Amyloid-Type Aggregation.

As a common pathological hallmark, protein aggregation into amyloids is a highly complicated phenomenon, attracting extensive research interest for elucidating its structural details and formation mechanisms. Membrane deposition and disulfide-driven protein misfolding play critical roles in amyloid-type aggregation, yet the underlying molecular process remains unclear. Here, we employed sum frequency generation (SFG) vibrational spectroscopy to comprehensively investigate the remodeling process of lysozyme, as the model protein, into amyloid-type aggregates at the cell membrane interface. It was discovered that disulfide reduction concurrently induced the transition of membrane-bound lysozyme from predominantly α-helical to antiparallel β-sheet structures, under a mode switch of membrane interaction from electrostatic to hydrophobic, and subsequent oligomeric aggregation. These findings shed light on the systematic understanding of dynamic molecular mechanisms underlying membrane-interactive amyloid oligomer formation.

Understanding the Structural Ballet of Cellulose Formation – Using CryoEM to Reveal the Role of Protein Flexibility on Higher-order Oligomer Formation, Glucan Catalysis, and Cellulose Microfibril Extrusion

Understanding the Structural Ballet of Cellulose Formation – Using CryoEM to Reveal the Role of Protein Flexibility on Higher-order Oligomer Formation, Glucan Catalysis, and Cellulose Microfibril Extrusion

Rosmarinic acid turned α-syn oligomers into non-toxic species preserving microtubules in Raw 264.7 cells

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the therapeutic is focused on several approaches including the inhibition of fibril formation by small compounds, avoiding the formation of cytotoxic oligomers. Thus, we decided to explore the capacity of compounds carrying catechol moieties to inhibit the progression of α-synuclein. Overall, the compounds rosmarinic acid (1), carnosic acid (2), carnosol (3), epiisorosmanol (4), and rosmanol (5) avoid the progression of fibril formation assessed by Thiofavine T (ThT), and atomic force microscopy images showed that morphology is influenced for the actions of compounds over fibrillization. Moreover, ITC experiments showed a Kd varying from 28 to 51 µM, the ΔG showed that the reaction between compounds and α-syn is spontaneous, and ΔH is associated with an exothermic reaction, suggesting the interactions of hydrogen bonds among compounds and α-syn. Docking experiments reinforce this idea showing the intermolecular interactions are mostly hydrogen bonding within the sites 2, 9, and 3/13 of α-synuclein, and compounds 1 and 5. Thus, compound 1, rosmarinic acid, interestingly interacts better with site 9 through catechol and Lysines. In cultured Raw 264. 7 cells, the presence of compounds showed that most of them can promote cell differentiation, especially rosmarinic acid, and rosmanol, both preserving tubulin cytoskeleton. However, once we evaluated whether or not the aggregates pre-treated with compounds could prevent the disruption of microtubules of Raw 264.7 cells, only pre-treated aggregates with rosmarinic acid prevented the disruption of the cytoskeleton. Altogether, we showed that especially rosmarinic acid not only inhibits α-syn but stabilizes the remaining aggregates turning them into not-toxic to Raw 264.7 cells suggesting a main role in cell survival and antigen processing in response to external α-syn aggregates.

Direct evidence for the mechanism of early-stage geopolymerization process

Geopolymer, an alternative construction material to cement, acquires strength and endurance through the geopolymerization process. However, the study on the early-stage geopolymerization is rare since it occurs rapidly and involves liquid phase, viscous slurry, and gel-like material, making the observation of this process complicated. This research addresses the challenge of observing this process by providing real-time evidence through in situ techniques: Fourier-transformed infrared (FTIR) spectroscopy, X-ray absorption spectroscopy (XAS), and environmental scanning electron microscopy (E-SEM). These methods consistently confirm the formation of aluminosilicate oligomers, short chains of silicate and aluminate species, in the polycondensation Stage I within the initial 5 minutes. This stage involves the reaction of Si-O and Al-O monomers, released during a dissolution process, with available Na2SiO3 from alkaline solutions in the system. In the subsequent polycondensation Stage II, aluminosilicate oligomers polymerize, creating a three-dimensional network of aluminosilicate chains through the crosslinking of Si-O-Si and Si-O-Al bonds. This process continues beyond the initial 5 minutes, gradually nearing completion around 30 minutes, as consistently confirmed by various experimental techniques. Rheological studies on the geopolymer paste flow rate support these interpretations. The study provides compelling direct evidence into the mechanisms of early-stage geopolymerization, significantly contributing to the research field and paving the way for widespread utilization in geopolymer applications.

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure.

Recent attention has focused on the de novo design of proteins, paralleling advancements in biopharmaceuticals. Achieving protein designs with both structure and function poses a significant challenge, particularly considering the importance of quaternary structures, such as oligomers, in protein function. The cell penetration properties of peptides are of particular interest as they involve the penetration of large molecules into cells. We previously suggested a link between the oligomerization propensity of amphipathic peptides and their cell penetration abilities, yet concrete evidence at cellular-relevant concentrations was lacking due to oligomers' instability. In this study, we sought to characterize oligomerization states using various techniques, including X-ray crystallography, acceptor photobleaching Förster resonance energy transfer (FRET), native mass spectrometry (MS), and differential scanning calorimetry (DSC), while exploring the function related to oligomer status. X-ray crystallography revealed the atomic structures of oligomers formed by LK-3, a bis-disulfide bridged dimer with amino acid sequence LKKLCLKLKKLCKLAG, and its derivatives, highlighting the formation of hexamers, specifically the trimer of dimers, which exhibited a stable hydrophobic core. FRET experiments showed that LK-3 oligomer formation was associated with cell penetration. Native MS confirmed higher-order oligomers of LK-3, while an intriguing finding was the enhanced cell-penetrating capability of a 1:1 mixture of l/d-peptide dimers compared to pure enantiomers. DSC analysis supported the notion that this enantiomeric mixture promotes the formation of functional oligomers, crucial for cell penetration. In conclusion, our study provides direct evidence that amphipathic peptide LK-3 forms oligomers at low nanomolar concentrations, underscoring their significance in cell penetration behavior.

Template-based copying in chemically fuelled dynamic combinatorial libraries

One of science’s greatest challenges is determining how life can spontaneously emerge from a mixture of molecules. A complicating factor is that life and its molecules are inherently unstable—RNA and proteins are prone to hydrolysis and denaturation. For the de novo synthesis of life or to better understand its emergence at its origin, selection mechanisms are needed for unstable molecules. Here we present a chemically fuelled dynamic combinatorial library to model RNA oligomerization and deoligomerization and shine new light on selection and purification mechanisms under kinetic control. In the experiments, oligomers can only be sustained by continuous production. Hybridization is a powerful tool for selecting unstable molecules, offering feedback on oligomerization and deoligomerization rates. Moreover, we find that templation can be used to purify libraries of oligomers. In addition, template-assisted formation of oligomers within coacervate-based protocells changes its compartment’s physical properties, such as their ability to fuse. Such reciprocal coupling between oligomer production and physical properties is a key step towards synthetic life.

Oligomer Formation Effects on the Separation of Trivalent Lanthanide Fission Products.

The assessment of trivalent lanthanide yields from the fission of uranium-235 is currently achieved using LN (LaNthanide) resin, di(2-ethylhexyl)orthophosphoric acid immobilized on a solid support. However, coelution of lighter lanthanides into terbium (Tb3+) fractions remains a significant problem in recovery of analytically pure fractions. In order to understand how the separation of trivalent lanthanides and yttrium (Ln3+) with LN resin proceeds and how to improve it, their speciation with the organic extractant HDEHP must be fully understood under aqueous conditions. A comprehensive luminescence analysis of aqueous solutions of Ln3+ in contact with HDEHP, along with infrared spectroscopy, elemental combustion analysis, inductively coupled plasma atomic emission spectroscopy (ICP-AES), and mass spectrometry, was used to indicate that an intermediate species is responsible for the coelution; where similar Ln3+ centers (e.g., Eu3+ and Tb3+) are bridged by the O-P-O moiety of deprotonated HDEHP to form large heteronuclear oligomeric structures with the general formula [Ln2(DEHP)6]n. Energy transfer from Tb3+ to Eu3+ in this structure confirms that lanthanide centers are within 10 Å and was used to propose that the oligomeric [Ln2(DEHP)6]n structure is formed rather than a dimeric Ln2(DEHP)6 structure. The effect of this speciation on LN resin column elution is investigated using luminescence spectroscopy, confirming that the oligomeric [Ln2(DEHP)6]n species could disrupt regular elution behavior and cause the problematic bleeding of lighter lanthanides (Sm3+ and Eu3+) into Tb3+ fractions. Resin luminescence measurements were used to propose that the bleeding of the organic extractant HDEHP from its solid support causes the formation of the disruptive oligometallic species.

Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

Leveraging Machine Learning-Guided Molecular Simulations Coupled with Experimental Data to Decipher Membrane Binding Mechanisms of Aminosterols.

Understanding the molecular mechanisms of the interactions between specific compounds and cellular membranes is essential for numerous biotechnological applications, including targeted drug delivery, elucidation of the drug mechanism of action, pathogen identification, and novel antibiotic development. However, estimation of the free energy landscape associated with solute binding to realistic biological systems is still a challenging task. In this work, we leverage the Time-lagged Independent Component Analysis (TICA) in combination with neural networks (NN) through the Deep-TICA approach for determining the free energy associated with the membrane insertion processes of two natural aminosterol compounds, trodusquemine (TRO), and squalamine (SQ). These compounds are particularly noteworthy because they interact with the outer layer of neuron membranes, protecting them from the toxic action of misfolded proteins involved in neurodegenerative disorders, in both their monomeric and oligomeric forms. We demonstrate how this strategy could be used to generate an effective collective variable for describing solute absorption in the membrane and for estimating free energy landscape of translocation via on-the-fly probability enhanced sampling (OPES) method. In this context, the computational protocol allowed an exhaustive characterization of the aminosterol entry pathway into a neuron-like lipid bilayer. Furthermore, it provided accurate prediction of membrane binding affinities, in close agreement with the experimental binding data obtained by using fluorescently labeled aminosterols and large unilamellar vesicles (LUVs). The findings contribute significantly to our understanding of aminosterol entry pathways and aminosterol-lipid membrane interactions. Finally, the computational methods deployed in this study further demonstrate considerable potential for investigating membrane binding processes.

Synaptotagmin-1 undergoes phase separation to regulate its calcium-sensitive oligomerization.

Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.