RESUMO
Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.
Assuntos
Bicamadas Lipídicas , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Humanos , Ligação Proteica , Multimerização ProteicaRESUMO
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 elevated concentrations, making insufficient solubility a critical bottleneck in the drug development pipeline. 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 µg of protein (>10-fold improvement), and yields 10,000 data points (1000-fold improvement). This allows quantitative comparison of formulation excipients, such as sodium chloride, polysorbate, histidine, arginine, and sucrose. Additionally, we can measure how solubility is affected by the combinatorial effect of multiple additives, find a suitable pH for the formulation, and measure the impact of mutations on solubility, thus enabling the screening of large libraries. By reducing material and time costs, this approach makes detailed multidimensional solubility optimization experiments possible, streamlining drug development and increasing our understanding of biotherapeutic solubility and the effects of excipients.
Assuntos
Excipientes , Microfluídica , Solubilidade , Polissorbatos , ProteínasRESUMO
Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.
RESUMO
A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of ß-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure-function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.
Assuntos
Mutação , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/genética , Humanos , Doenças Neurodegenerativas , Agregados Proteicos , Ligação Proteica , Multimerização Proteica/genética , Análise Espectral , alfa-Sinucleína/metabolismoRESUMO
The formation of ordered protein and peptide assemblies is a phenomenon related to a wide range of human diseases. However, the mechanism of assembly at the molecular level remains largely unknown. Minimal models enable the exploration of the underlying interactions that are at the core of such self-assembly processes. In particular, the ability of phenylalanine, a single aromatic amino acid, to form an amyloid-like structure has challenged the previous dogma viewing a peptide backbone as a prerequisite for assembly. The driving forces controlling the nucleation and assembly in the absence of a peptide backbone remain to be identified. Here, aiming to unravel these forces, we explored the kinetics and thermodynamics of three phenylalanine-containing molecules during their assembly process: the amino acid phenylalanine, which accumulates in phenylketonuria patients, the diphenylalanine core-motif of the amyloid ß peptide related to Alzheimer's disease, and the extended triphenylalanine peptide which forms a range of distinct nanostructures in vitro. We found that the aggregation propensity, regarding the critical monomer concentration, strongly increases with size, with triphenylalanine being the most aggregation-prone species under our experimental conditions. In the context of classical nucleation theory, this increase in aggregation propensity can be attributed to the larger free energy decrease upon aggregation of larger peptides and is not due to the presence/absence of a peptide bond per se. Taken together, this work provides insights into the aggregation processes of chemically simple systems and suggests that both backbone-containing peptides and backbone-lacking amino acids assemble through a similar mechanism, thus supporting the classification of amino acids in the continuum of amyloid-forming building blocks.
Assuntos
Peptídeos beta-Amiloides , Fenilalanina , Humanos , Peptídeos beta-Amiloides/química , Termodinâmica , Fenilalanina/química , Cinética , Amiloide/químicaRESUMO
The abundance of plant-derived proteins, as well as their biodegradability and low environmental impact make them attractive polymeric feedstocks for next-generation functional materials to replace current petroleum-based systems. However, efforts to generate functional materials from plant-based proteins in a scalable manner have been hampered by the lack of efficient methods to induce and control their micro and nanoscale structure, key requirements for achieving advantageous material properties and tailoring their functionality. Here, we demonstrate a scalable approach for generating mechanically robust plant-based films on a metre-scale through controlled nanometre-scale self-assembly of water-insoluble plant proteins. The films produced using this method exhibit high optical transmittance, as well as robust mechanical properties comparable to engineering plastics. Furthermore, we demonstrate the ability to impart nano- and microscale patterning into such films through templating, leading to the formation of hydrophobic surfaces as well as structural colour by controlling the size of the patterned features.
Assuntos
Nanoestruturas/química , Proteínas de Plantas/química , Polímeros/química , Propriedades de Superfície , Hidrogéis/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanoestruturas/ultraestrutura , Solubilidade , Solventes/química , Glycine max/química , Glycine max/metabolismo , Água/químicaRESUMO
Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.
Assuntos
Peptídeos , Cápsulas , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Proteins are the fundamental building blocks for high-performance materials in nature. Such materials fulfill structural roles, as in the case of silk and collagen, and can generate active structures including the cytoskeleton. Attention is increasingly turning to this versatile class of molecules for the synthesis of next-generation green functional materials for a range of applications. Protein nanofibrils are a fundamental supramolecular unit from which many macroscopic protein materials are formed. In this Review, we focus on the multiscale assembly of such protein nanofibrils formed from naturally occurring proteins into new supramolecular architectures and discuss how they can form the basis of material systems ranging from bulk gels, films, fibers, micro/nanogels, condensates, and active materials. We review current and emerging approaches to process and assemble these building blocks in a manner which is different to their natural evolutionarily selected role but allows the generation of tailored functionality, with a focus on microfluidic approaches. We finally discuss opportunities and challenges for this class of materials, including applications that can be involved in this material system which consists of fully natural, biocompatible, and biodegradable feedstocks yet has the potential to generate materials with performance and versatility rivalling that of the best synthetic polymers.
Assuntos
Materiais Biocompatíveis , Seda , GéisRESUMO
Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.
Assuntos
Lipídeos/química , Emulsões/síntese química , Emulsões/química , Bicamadas Lipídicas/química , Óleos/química , Tamanho da Partícula , Propriedades de Superfície , Água/químicaRESUMO
The detection and analysis of proteins in a label-free manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 µg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.
Assuntos
Técnicas Biossensoriais , Cromatografia Líquida , Técnicas de Microbalança de Cristal de Quartzo , Proteína Estafilocócica ARESUMO
Correction for 'Correction: Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions' by Zenon Toprakcioglu et al., Soft Matter, 2020, 16, 3586-3586, DOI: .
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Membrane-less organelles resulting from liquid-liquid phase separation of biopolymers into intracellular condensates control essential biological functions, including messenger RNA processing, cell signalling and embryogenesis1-4. It has recently been discovered that several such protein condensates can undergo a further irreversible phase transition, forming solid nanoscale aggregates associated with neurodegenerative disease5-7. While the irreversible gelation of protein condensates is generally related to malfunction and disease, one case where the liquid-to-solid transition of protein condensates is functional, however, is that of silk spinning8,9. The formation of silk fibrils is largely driven by shear, yet it is not known what factors control the pathological gelation of functional condensates. Here we demonstrate that four proteins and one peptide system, with no function associated with fibre formation, have a strong propensity to undergo a liquid-to-solid transition when exposed to even low levels of mechanical shear once present in their liquid-liquid phase separated form. Using microfluidics to control the application of shear, we generated fibres from single-protein condensates and characterized their structural and material properties as a function of shear stress. Our results reveal generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. These observations suggest that shear can play an important role in the irreversible liquid-to-solid transition of protein condensates, shed light on the role of physical factors in driving this transition in protein aggregation-related diseases and open a new route towards artificial shear responsive biomaterials.
Assuntos
Peptídeos/química , Transição de Fase , Proteínas/química , Animais , Fenômenos Biomecânicos , Bombyx/química , Linhagem Celular , Fibroínas/química , Agregados Proteicos , Estresse Mecânico , Resistência à Tração , TermodinâmicaRESUMO
The biological function of proteins is dictated by the formation of supra-molecular complexes that act as the basic machinery of the cell. As such, measuring the properties of protein species in heterogeneous mixtures is of key importance for understanding the molecular basis of biological function. Here, we describe the combination of analytical microfluidic tools with liquid chromatography for multidimensional characterisation of biomolecules in complex mixtures in the solution phase. Following chromatographic separation, a small fraction of the flow-through is distributed to multiple microfluidic devices for analysis. The microfluidic device developed here allows the simultaneous determination of the hydrodynamic radius, electrophoretic mobility, effective molecular charge and isoelectric point of isolated protein species. We demonstrate the operation principle of this approach with a mixture of three unlabelled model proteins varying in size and charge. We further extend the analytical potential of the presented approach by analysing a mixture of interacting streptavidin with biotinylated BSA and fluorophores, which form a mixture of stable complexes with diverse biophysical properties and stoichiometries. The presented microfluidic device positioned in-line with liquid chromatography presents an advanced tool for characterising multidimensional physical properties of proteins in biological samples to further understand the assembly/disassembly mechanism of proteins and the nature of complex mixtures.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Proteínas , Eletroforese , Dispositivos Lab-On-A-Chip , Proteínas/análiseRESUMO
Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.
Assuntos
Endopeptidase K/química , Glucose/análise , Técnicas Analíticas Microfluídicas , Dióxido de Silício/química , Endopeptidase K/metabolismo , Humanos , Tamanho da Partícula , Dióxido de Silício/metabolismo , Propriedades de SuperfícieRESUMO
Exploration and characterisation of the human proteome is a key objective enabling a heightened understanding of biological function, malfunction and pharmaceutical design. Since proteins typically exhibit their behaviour by binding to other proteins, the challenge of probing protein-protein interactions has been the focus of new and improved experimental approaches. Here, we review recently developed microfluidic techniques for the study and quantification of protein-protein interactions. We focus on methodologies that utilise the inherent strength of microfluidics for the control of mass transport on the micron scale, to facilitate surface and membrane-free interrogation and quantification of interacting proteins. Thus, the microfluidic tools described here provide the capability to yield insights on protein-protein interactions under physiological conditions. We first discuss the defining principles of microfluidics, and methods for the analysis of protein-protein interactions that utilise the diffusion-controlled mixing characteristic of fluids at the microscale. We then describe techniques that employ electrophoretic forces to manipulate and fractionate interacting protein systems for their biophysical characterisation, before discussing strategies that use microdroplet compartmentalisation for the analysis of protein interactions. We conclude by highlighting future directions for the field, such as the integration of microfluidic experiments into high-throughput workflows for the investigation of protein interaction networks.
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Correction for 'Multi-scale microporous silica microcapsules from gas-in water-in oil emulsions' by Zenon Toprakcioglu et al., Soft Matter, 2020, DOI: 10.1039/c9sm02274k.
RESUMO
Controlling the surface area, pore size and pore volume of microcapsules is crucial for modulating their activity in applications including catalytic reactions, delivery strategies or even cell culture assays, yet remains challenging to achieve using conventional bulk techniques. Here we describe a microfluidics-based approach for the formation of monodisperse silica-coated micron-scale porous capsules of controllable sizes. Our method involves the generation of gas-in water-in oil emulsions, and the subsequent rapid precipitation of silica which forms around the encapsulated gas bubbles resulting in hollow silica capsules with tunable pore sizes. We demonstrate that by varying the gas phase pressure, we can control both the diameter of the bubbles formed and the number of internal bubbles enclosed within the silica microcapsule. Moreover, we further demonstrate, using optical and electron microscopy, that these silica capsules remain stable under particle drying. Such a systematic manner of producing silica-coated microbubbles and porous microparticles thus represents an attractive class of biocompatible material for biomedical and pharmaceutical related applications.
Assuntos
Cápsulas/química , Emulsões/química , Óleos/química , Dióxido de Silício/química , Água/química , Materiais Biocompatíveis/química , Composição de Medicamentos/instrumentação , Desenho de Equipamento , Gases/química , Microbolhas , PorosidadeRESUMO
Microemulsions have found a wide range of applications exploiting their chemical and physical properties. Development of microfluidic-based approaches has allowed for the controlled production of highly monodispersed emulsions, including the formation of multiple and hierarchical emulsions. Conventional poly(dimethylsiloxane)-based microfluidic systems require tight spatial control over the surface chemistry when used for double emulsion generation, which can be challenging to achieve on the micrometer scale. Here, we present a two-dimensional device design, which can selectively be surface-treated in a straightforward manner and allows for the formation of uniform water/oil/water double emulsions by combining two distinct hydrophilic and hydrophobic surface properties. These surfaces are sufficiently separated in space to allow for imparting their functionalization without the requirement for lithographic approaches or complex flow control. We demonstrate that a mismatch between the wettability requirements of the continuous phase and the channel wall inherent in this approach can be tolerated over several hundreds of micrometers, opening up the possibility to use simple pressure-driven flows to achieve surface functionalization. The design architecture exhibits robust efficiency in emulsion generation while retaining simple device fabrication. We finally demonstrate the potential of this approach by generating water in oil in water emulsions with lipid molecules acting as surfactants.
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Self-assembling peptides and proteins have the potential to serve as multifunctional building blocks for the generation of versatile materials for a wide range of biomedical applications. In particular, supramolecular hydrogels comprised of self-assembled protein nanofibrils, have been used in contexts ranging from tissue engineering to drug delivery. Due to the rapid emergence of multidrug resistant bacteria, development of biomaterials with intrinsic antimicrobial properties has been continuously increasing. Here, we describe hybrid organic/inorganic nanofibrillar silk microgels decorated with silver nanoparticles that display potent antimicrobial activity in vitro and in vivo and are able to adhere bacterial cells to their surfaces while subsequently eradicating them, through a two-step mechanism of action. Importantly, in contrast to treatments involving conventional silver, these silk-silver microgels are nonhemolytic and noncytotoxic toward mammalian cell lines. Finally, we show that these hybrid microgels display substantial efficacy as topical antimicrobial agents in a murine model of surgical site infections.
Assuntos
Antibacterianos , Anti-Infecciosos , Bactérias/crescimento & desenvolvimento , Aderência Bacteriana/efeitos dos fármacos , Infecções Bacterianas , Hidrogéis , Nanopartículas Metálicas , Nanofibras , Seda , Prata , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Hidrogéis/química , Hidrogéis/farmacologia , Teste de Materiais , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Nanofibras/química , Nanofibras/uso terapêutico , Ovinos , Seda/química , Seda/farmacologia , Prata/química , Prata/farmacologiaRESUMO
Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.