Papers by Tomas Šneideris
Proceedings of the National Academy of Sciences
Nonspecific interactions are a key challenge in the successful development of therapeutic antibod... more Nonspecific interactions are a key challenge in the successful development of therapeutic antibodies. The tendency for nonspecific binding of antibodies is often difficult to reduce by rational design, and instead, it is necessary to rely on comprehensive screening campaigns. To address this issue, we performed a systematic analysis of the impact of surface patch properties on antibody nonspecificity using a designer antibody library as a model system and single-stranded DNA as a nonspecificity ligand. Using an in-solution microfluidic approach, we find that the antibodies tested bind to single-stranded DNA with affinities as high as K D = 1 µM. We show that DNA binding is driven primarily by a hydrophobic patch in the complementarity-determining regions. By quantifying the surface patches across the library, the nonspecific binding affinity is shown to correlate with a trade-off between the hydrophobic and total charged patch areas. Moreover, we show that a change in formulation co...
Communications biology, Jan 4, 2021
Aberrant soluble oligomers formed by the amyloid-β peptide (Aβ) are major pathogenic agents in th... more Aberrant soluble oligomers formed by the amyloid-β peptide (Aβ) are major pathogenic agents in the onset and progression of Alzheimer's disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aβ aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aβ oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aβ aggregation and key biochemical processes regulating cellular homeostasis in the brain.
bioRxiv (Cold Spring Harbor Laboratory), Mar 7, 2022
Non-specificity is a key challenge in the successful development of therapeutic antibodies. The t... more 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. 1
bioRxiv (Cold Spring Harbor Laboratory), Jun 13, 2023
Binary phase separation with conserved order parameters is a generic thermodynamic process that a... more Binary phase separation with conserved order parameters is a generic thermodynamic process that arises in different contexts. One system of great fundamental and practical interest is the condensation of bio-molecules, where the order parameters are concentrations of solutes, and the condensed phase typically contains many different components of diverse properties. In such systems, each solute species corresponds to one dimension in the full phase space and a major challenge in the field is to rigorously interpret and quantify the multi-component character of any given phase separation system. Here, we formulate a dominance parameter D α for each solute α in a multicomponent system, and show that it is easily accessible in experiments and relates to the underlying thermodynamics and the phase space. We complement the theoretical work with experimental data, and demonstrate that D α reflects the interactions driving phase separation. A simple computational model further illuminates the variation of D α across the whole phase space. This work paves the way for future quantitative investigations into multi-component systems.
Biophysical Journal, Feb 1, 2023
bioRxiv (Cold Spring Harbor Laboratory), Oct 22, 2022
Protein-based biologics are highly suitable for drug development, as they exhibit low toxicity an... more 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 ultrahigh-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.
Proceedings of the National Academy of Sciences of the United States of America, Jun 3, 2020
International Journal of Molecular Sciences, Feb 10, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Physical Chemistry B, Oct 1, 2021
In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy ... more In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aβ(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a β-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended β-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aβ(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aβ(1-42) structures. The calculations showed that only structures with a significant amyloid β-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.
Physical Chemistry Chemical Physics, 2018
bioRxiv (Cold Spring Harbor Laboratory), Jun 3, 2023
Biomolecular phase separation has emerged as a critical process for regulating cellular organisat... more Biomolecular phase separation has emerged as a critical process for regulating cellular organisation and function. Modulators, ranging from ionic species to small molecule compounds and macromolecular binders, are key in controlling phase behaviour. The mechanisms of action of such modulators, however, have remained challenging to resolve as means to study protein-modulator interactions within condensates are lacking. Here we unravel biophysical mechanisms of protein phase separation modulation by applying a tie-line-gradientbased analysis approach to characterize the effects of modulators on the partitioning and interactions of species across the dense and dilute phases. Using the protein fused in sarcoma (FUS), we first characterize the impact of ionic species on FUS phase separation and find that potassium chloride (KCl) ions are preferentially excluded from condensates at low ionic strengths as they counteract intermolecular interactions by charge screening. Conversely, in the high salt reentrant phase separation regime, salts preferentially partition into the dense phase thereby scaffolding condensates by enhancing non-ionic interactions. Furthermore, we show that the common phase separation disruptor 1,6-hexanediol decreases FUS phase separation propensity by acting as a solvation agent for the protein. This leads to an expansion of the polypeptide chain, which limits the ability of FUS to form inter-and intramolecular interactions, thereby counteracting condensation. Lastly, we study a sequence-specific modulator of FUS phase separation using a FUS-targeting RNA aptamer and find a concentration-dependent response of condensate stabilisation already at nanomolar concentrations. The sequence-specific binder first partitions as a client molecule into the condensate and then acts as a scaffolder within the condensate. Taken together, our study highlights a broadly applicable approach for studying condensate partitioning allowing us to decipher the mechanisms of action of phase separation modulators.
Advanced Functional Materials, Apr 15, 2019
bioRxiv (Cold Spring Harbor Laboratory), Jan 13, 2023
bioRxiv (Cold Spring Harbor Laboratory), Feb 10, 2022
The formation of biomolecular condensates through liquid-liquid phase separation from proteins an... more 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.
Biomolecules, Dec 11, 2019
Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's ... more Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clinical trials is very high, even for compounds that show promising anti-amyloid activity in vitro. In this study, we demonstrate that even small changes in the chemical environment can strongly modulate the inhibitory effects of anti-amyloid compounds. Using one of the best-established amyloid inhibitory compounds, epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related α-synuclein, we shed light on the previously unexplored sensitivity to solution conditions of the action of this compound on amyloid fibril formation. In the case of insulin, we show that the classification of EGCG as an amyloid inhibitor depends on the experimental conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the experiment. For α-synuclein, we show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behaviour by the increased stability of EGCG against oxidation at pH 6.
PeerJ, Aug 16, 2019
Protein aggregation into amyloid fibrils has been linked to multiple neurodegenerative disorders.... more Protein aggregation into amyloid fibrils has been linked to multiple neurodegenerative disorders. Determining the kinetics of fibril formation, as well as their structural stability are important for the mechanistic understanding of amyloid aggregation. Tracking both fibril association and dissociation is usually performed by measuring light scattering of the solution or fluorescence of amyloid specific dyes, such as thioflavin-T. A possible addition to these methods is the recently discovered deep-blue autofluorescence (dbAF), which is linked to amyloid formation. In this work we explore the potential of this phenomenon to monitor amyloid fibril formation and dissociation, as well as show its possible relation to fibril size rather than amyloid structure.
SUMMARYPhase transitions of cellular proteins and lipids play a key role in governing the organis... more SUMMARYPhase 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 o...
Antimicrobial peptides (AMPs) are a vital part of the immune system that helps combat bacterial i... more 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 o...
International Journal of Molecular Sciences, 2021
Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of ho... more Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein’s fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.
Communications Biology, 2021
A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-01680-7.
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Papers by Tomas Šneideris