Biology subjects

Sneideris, T.

Publications and source records attributed to Sneideris, T..

11 recordsLinked to original sources

bioRxiv2024

Temperature-induced changes in protein interactions control RNA recruitment to G3BP1 condensates

Biomolecular condensates have emerged as prominent regulators of dynamic subcellular organisation and essential biological processes. Temperature, in particular, exerts a significant influence on the formation and behaviour of biomolecular condensation. For example, during cellular heat stress, stress granules (SGs) are formed from RNA-binding proteins (RBPs) and RNA, forming liquid condensates to protect the RNA from damage. However, the molecular mechanisms leading to changes in protein phase behaviour are not well understood. To answer how temperature modulates protein interactions and phase behaviour, we developed a high-throughput microfluidic platform, capable of mapping the phase space and quantifying protein interactions in a temperature-dependent manner. Specifically, our approach measures high-resolution protein phase diagrams as a function of temperature, while accurately quantifying changes in the binodal, condensate stoichiometry and free energy contribution of a solute, hence, providing information about the underlying mechanistic driving forces. We employ this approach to investigate the effect of temperature changes on the phase separation of the stress granule scaffold protein Ras GTPase-activating protein-binding protein 1 (G3BP1) with PolyA-RNA. Surprisingly, we find that the G3BP1/RNA phase boundary remains unaffected by the increasing temperature but the underlying stoichiometry and energetics shift, which can only be revealed with high-resolution phase diagrams. This indicates that temperature-induced dissolution is counteracted by entropic processes driving phase separation. With increasing temperature, the G3BP1 content in condensates decreases alongside with a reduction of the free energy of protein interactions, while the RNA content increases driven by entropically favoured hydrophobic interactions. In the context of cellular heat SG formation, these findings could indicate that during heat shock, elevated temperatures directly induce RNA recruitment to stress granules as a cytoprotective mechanism by finetuning the strength of protein and RNA interactions.

biophysics↗

bioRxiv2024

The Alzheimer's Aβ peptide forms biomolecular condensates that trigger amyloid aggregation

The onset and development of Alzheimers disease (AD) is linked to the accumulation of pathological aggregates formed from the normally monomeric amyloid-{beta} peptide within the central nervous system. These A{beta} aggregates are increasingly successfully targeted with clinical therapies, but the fundamental molecular steps that trigger the initial nucleation event leading to the conversion of monomeric A{beta} peptide into pathological aggregates remain unknown. Here we show that the A{beta} peptide can form biomolecular condensates on lipid bilayers both in molecular assays and in living cells. Our results reveal that these A{beta} condensates can significantly accelerate the primary nucleation step in the amyloid conversion cascade that leads to the formation of amyloid aggregates and plaque. We show that A{beta} condensates contain phospholipids, are intrinsically heterogenous, and are prone to undergo a liquid-to-solid transition leading to the formation amyloid fibrils. These findings uncover the liquid-liquid phase separation behaviour of the A{beta} peptide, and reveal a new molecular step very early in the amyloid-{beta} aggregation cascade that can form the basis for novel therapeutic intervention strategies. Significance statementThe hallmark of Alzheimers disease is the abnormal buildup of the normally soluble amyloid {beta} protein aggregates in the central nervous system. While the molecular mechanisms at the late stages of the amyloid {beta} aggregation cascade are well understood, the initial steps remained elusive until now. Our current study demonstrates that amyloid {beta} undergoes liquid-liquid phase separation on lipid surfaces, which triggers primary nucleation and initiates the amyloid {beta} aggregation cascade. This newly identified step in the molecular mechanism of Alzheimers disease represents a promising target for the development of alternative innovative therapeutic strategies.

biophysics↗

bioRxiv2023

Linking modulation of bio-molecular phase behaviour with collective interactions

Bio-molecular condensates formed in the cytoplasm of cells are increasingly recognised as key spatiotemporal organisers of living matter, and are implicated in a wide range of functional or pathological processes. This discovery opens up a new avenue for condensate-based applications and a crucial step in controlling this process is to understand the underlying interactions driving condensate formation or dissolution. However, these condensates are highly multi-component assemblies and many inter-component interactions are present, rendering it difficult to identify key promoters of phase separation. In this work, we extend the recently formulated dominance analysis to modulations of condensate formation. By carrying out dilute phase concentration measurements of a single target solute, the theoretical framework allows one to deduce whether the modulator acts on the target solute or another unspecified, auxiliary solute, as well as the attractive/repulsive nature of the added interaction. This serve as a general guide towards deducing possible modulation mechanisms on the molecular level, which can be complemented by orthogonal measurements. As a case study, we investigate the modulation of G3BP1/RNA condensates by the small molecule suramin, and the dominance measurements point towards a dissolution mechanism where suramin acts on G3BP1 to disrupt G3BP1/RNA interactions, as confirmed by a diffusional sizing assay. Our approach and the dominance framework have a high degree of adaptability and can be applied in many other condensate-forming systems.

biophysics↗

bioRxiv2023

Dominance metric in multi-component binary phase equilibria

Phase separation in aqueous solutions of macromolecules is thought to underlie the generation of biomolecular condensates in cells. Condensates are membraneless bodies, representing dense, macromolecule-rich phases that coexist with the dilute, macromolecule-deficient phase. In cells, condensates comprise hundreds of different macromolecular and small molecule solutes. Do all components contribute equally or very differently to the driving forces for phase separation? Currently, we lack a coherent formalism to answer this question, a gap we remedy in this work through the introduction of a formalism we term energy dominance analysis. This approach rests on model-free analysis of shapes of the dilute arms of phase boundaries, slopes of tie lines, and changes to dilute phase concentrations in response to perturbations of concentrations of different solutes. We present the formalism that underlies dominance analysis, and establish its accuracy and flexibility by deploying it to analyse phase spaces probed in silico, in vitro, and in cellulo.

biophysics↗

bioRxiv2023

Condensate partitioning governs the mechanism of action of FUS phase separation modulators

Biomolecular phase separation plays a pivotal role in governing critical biological functions and arises from the collective interactions of large numbers of molecules. Characterising the underlying collective interactions of phase separation, however, has proven to be challenging with currently available tools. Here, we propose a general and easily accessible strategy to quantify collective interactions in biomolecular phase separation with respect to composition and energetics. By measuring the dilute phase concentration of one species only, we determine tie line gradients and free energy dominance as dedicated descriptors of collective interactions. We apply this strategy to dissect the role of salts and small molecules on phase separation of the protein fused in sarcoma (FUS). We discover that monovalent salts can display both exclusion from or preferential partitioning into condensates to either counteract charge screening or enhance non-ionic interactions. Moreover, we show that the common hydrophobic interaction disruptor 1,6-hexanediol inhibits FUS phase separation by acting as a solvation agent capable of expanding the protein polypeptide chain. Taken together, our work presents a widely applicable strategy that enables quantification of collective interactions and provides unique insights into the underlying mechanisms of condensate formation and modulation.

biophysics↗

bioRxiv2023

ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/533832v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@16f3e10org.highwire.dtl.DTLVardef@538f39org.highwire.dtl.DTLVardef@1e00d4eorg.highwire.dtl.DTLVardef@89967d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Modulating Nucleic Acid Phase Transitions as a Mechanism of Action for Cell-Penetrating Antimicrobial Peptides

Antimicrobial peptides (AMPs) are a vital part of the immune system that helps combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we have discovered that a significant number of AMPs have a strong tendency to form liquid-like condensates in the presence of nucleic acids, a process known as phase separation. We have demonstrated that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. We propose that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. These findings open up exciting possibilities for the development of antimicrobials that target nucleic acid phase transitions.

microbiology↗

bioRxiv2023

Adsorption of RNA to interfaces of biomolecular condensates enables wetting transitions

Biomolecular condensates form via spontaneous and driven phase transitions of multivalent proteins and nucleic acids. These macromolecules can be organized in spatially inhomogeneous ways that lead to multiple coexisting dense phases with distinct macromolecular interfaces. While considerable attention has focused on the physical driving forces that give rise to phase separation from bulk solutions, the interactions that underlie adsorption driven wetting transitions remain unclear. Here, we report that pyrimidine-rich RNAs function as adsorbents that enable cascades of wetting transitions that include partial and complete wetting of condensates formed by purine-rich RNAs. Computations show that macromolecules that are scaffolds of condensates are oriented perpendicular to condensate interfaces whereas adsorbents are oriented parallel to interfaces. Our results yield heuristics for the design of synthetic materials that can be based on RNA-rich condensates featuring bespoke interfaces and distinct local microenvironments created by the interplay between scaffolds versus adsorbents.

biophysics↗

bioRxiv2022

Multi-dimensional protein solubility optimization with an ultra-high-throughput microfluidic platform

Protein-based biologics are highly suitable for drug development, as they exhibit low toxicity and high specificity for their targets. However, for therapeutic applications, biologics must often be formulated to very high concentrations, making insufficient solubility a critical bottleneck in drug development pipelines. Here, we report an ultra-high-throughput microfluidic platform for protein solubility screening. In comparison with previous methods, this microfluidic platform can make, incubate, and measure samples in a few minutes, uses just 20 micrograms of protein (> 10-fold improvement) and yields 10,000 data points (1000-fold improvement). This allows quantitative comparison of formulation additives, such as salt, polysorbate, histidine, arginine and sucrose. Additionally, we can measure how solubility is affected by different concentrations of multiple additives, find a suitable pH for the formulation, and measure the impact of single mutations on solubility, thus enabling the screening of large libraries. By reducing material and time costs, this approach makes detailed multi-dimensional solubility optimization experiments possible, streamlining drug development and increasing our understanding of biotherapeutic solubility and the effects of excipients.

biophysics↗

bioRxiv2022

Surface interaction patches link non-specific binding and phase separation of antibodies

Non-specificity is a key challenge in the successful development of therapeutic antibodies. The tendency for non-specific binding in antibodies is often difficult to reduce via judicious design and, instead, it is necessary to rely on comprehensive screening campaigns. A better understanding of the molecular origins that drive antibody non-specificity is therefore highly desirable in order to prevent non-specific off-target binding. Here, we perform a systematic analysis of the impact of surface patch properties on antibody non-specificity using a designer antibody library as a model system and DNA as a non-specificity ligand. Using an in solution microfluidics approach, we discover patches of surface hydrogen bonding to be causative of the observed non-specificity under physiological salt conditions and suggest them to be a vital addition to the molecular origins of non-specificity. Moreover, we find that a change in formulation conditions leads to DNA-induced antibody liquid-liquid phase separation as a manifestation of antibody non-specificity. We show that this behaviour is driven by a cooperative electrostatic network assembly mechanism enabled by mutations that yield a positively charged surface patch. Together, our study provides a direct link between molecular binding events and macroscopic liquid-liquid phase separation. These findings highlight a delicate balance between surface interaction patches and their crucial role in conferring antibody non-specificity.

biophysics↗

bioRxiv2022

Multiphase condensates from a kinetically arrested phase transition

The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

biophysics↗