Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinson's disease pathology is the aggregation of the α-synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric α-synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of α-synuclein aggregation, their detailed mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a mechanistic model that is able to globally describe the aggregation behaviour of α-synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation as much by enabling fibril elongation as by enabling their initial formation. Moreover, we find that the primary nucleation of lipid-protein co-aggregates takes place not on the surface of lipid vesicles in bulk solution but at the air-water and/or plate interfaces, where lipids and proteins are likely adsorbed. Our model forms the basis for mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the α-synuclein aggregation cascade.

Pharmacological inhibition of α-synuclein aggregation within liquid condensates.

Aggregated forms of α-synuclein constitute the major component of Lewy bodies, the proteinaceous aggregates characteristic of Parkinson's disease. Emerging evidence suggests that α-synuclein aggregation may occur within liquid condensates formed through phase separation. This mechanism of aggregation creates new challenges and opportunities for drug discovery for Parkinson's disease, which is otherwise still incurable. Here we show that the condensation-driven aggregation pathway of α-synuclein can be inhibited using small molecules. We report that the aminosterol claramine stabilizes α-synuclein condensates and inhibits α-synuclein aggregation within the condensates both in vitro and in a Caenorhabditis elegans model of Parkinson's disease. By using a chemical kinetics approach, we show that the mechanism of action of claramine is to inhibit primary nucleation within the condensates. These results illustrate a possible therapeutic route based on the inhibition of protein aggregation within condensates, a phenomenon likely to be relevant in other neurodegenerative disorders.

Aß Oligomer Dissociation Is Catalyzed by Fibril Surfaces.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aß peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aß42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Solutes unmask differences in clustering versus phase separation of FET proteins.

Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Dominance Analysis: A formalism to uncover dominant energetic contributions to biomolecular condensate formation in multicomponent systems.

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 .

Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning.

Machine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.

Maximum entropy determination of mammalian proteome dynamics.

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Design of amyloidogenic peptide traps.

Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.

Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.

Maturation-dependent changes in the size, structure and seeding capacity of Aß42 amyloid fibrils.

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-ß network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-ß sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

A Relationship between the Structures and Neurotoxic Effects of Aß Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.

Fabrication of gradient hydrogels using a thermophoretic approach in microfluidics.

The extracellular matrix presents spatially varying physical cues that can influence cell behavior in many processes. Physical gradients within hydrogels that mimic the heterogenous mechanical microenvironment are useful to study the impact of these cues on cellular responses. Therefore, simple and reliable techniques to create such gradient hydrogels are highly desirable. This work demonstrates the fabrication of stiffness gradient Gellan gum (GG) hydrogels by applying a temperature gradient across a microchannel containing hydrogel precursor solution. Thermophoretic migration of components within the precursor solution generates a concentration gradient that mirrors the temperature gradient profile, which translates into mechanical gradients upon crosslinking. Using this technique, GG hydrogels with stiffness gradients ranging from 20 to 90 kPa over 600µm are created, covering the elastic moduli typical of moderately hard to hard tissues. MC3T3 osteoblast cells are then cultured on these gradient substrates, which exhibit preferential migration and enhanced osteogenic potential toward the stiffest region on the gradient. Overall, the thermophoretic approach provides a non-toxic and effective method to create hydrogels with defined mechanical gradients at the micron scale suitable forin vitrobiological studies and potentially tissue engineering applications.

Self-replication of Aß42 aggregates occurs on small and isolated fibril sites.

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer's A[Formula: see text] amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A[Formula: see text] fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

Selenium-silk microgels as antifungal and antibacterial agents.

Antimicrobial resistance is a leading threat to global health. Alternative therapeutics to combat the rise in drug-resistant strains of bacteria and fungi are thus needed, but the development of new classes of small molecule therapeutics has remained challenging. Here, we explore an orthogonal approach and address this issue by synthesising micro-scale, protein colloidal particles that possess potent antimicrobial properties. We describe an approach for forming silk-based microgels that contain selenium nanoparticles embedded within the protein scaffold. We demonstrate that these materials have both antibacterial and antifungal properties while, crucially, also remaining highly biocompatible with mammalian cell lines. By combing the nanoparticles with silk, the protein microgel is able to fulfill two critical functions; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while simultaneously serving as a carrier for microbial eradication. Furthermore, since the antimicrobial activity originates from physical contact, bacteria and fungi are unlikely to develop resistance to our hybrid biomaterials, which remains a critical issue with current antibiotic and antifungal treatments. Therefore, taken together, these results provide the basis for innovative antimicrobial materials that can target drug-resistant microbial infections.

Nonspecificity fingerprints for clinical-stage antibodies in solution.

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs does not solely rely on optimal potency and safety but also require good biophysical properties to ensure a high developability potential. In particular, nonspecific interactions are a key developability parameter to monitor during discovery and development. Despite an increased focus on the detection of nonspecific interactions, their underlying physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterize a set of clinical-stage mAbs and their interactions with commonly used nonspecificity ligands to generate nonspecificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to measure binding affinities directly, and we evaluate the contribution of avidity in nonspecific binding by mAbs. We find that avidity can increase the apparent affinity by two orders of magnitude. Notably, we find that a subset of these highly developed mAbs show nonspecific electrostatic interactions, even at physiological pH and ionic strength, and that they can form microscale particles with charge-complementary polymers. The group of mAb constructs flagged here for nonspecificity are among the worst performers in independent reports of surface and column-based screens. The solution measurements improve on the state-of-the-art by providing a stand-alone result for individual mAbs without the need to benchmark against cohort data. Based on our findings, we propose a quantitative solution-based nonspecificity score, which can be integrated in the development workflow for biological therapeutics and more widely in protein engineering.

Targeting nucleic acid phase transitions as a mechanism of action for antimicrobial peptides.

Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate 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. These results suggest 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. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

Effects of Processing-Induced Contamination on Organic Electronic Devices.

Organic semiconductors are a family of pi-conjugated compounds used in many applications, such as displays, bioelectronics, and thermoelectrics. However, their susceptibility to processing-induced contamination is not well understood. Here, it is shown that many organic electronic devices reported so far may have been unintentionally contaminated, thus affecting their performance, water uptake, and thin film properties. Nuclear magnetic resonance spectroscopy is used to detect and quantify contaminants originating from the glovebox atmosphere and common laboratory consumables used during device fabrication. Importantly, this in-depth understanding of the sources of contamination allows the establishment of clean fabrication protocols, and the fabrication of organic field effect transistors (OFETs) with improved performance and stability. This study highlights the role of unintentional contaminants in organic electronic devices, and demonstrates that certain stringent processing conditions need to be met to avoid scientific misinterpretation, ensure device reproducibility, and facilitate performance stability. The experimental procedures and conditions used herein are typical of those used by many groups in the field of solution-processed organic semiconductors. Therefore, the insights gained into the effects of contamination are likely to be broadly applicable to studies, not just of OFETs, but also of other devices based on these materials.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.