Spatial control of irreversible protein aggregation

Liquid cellular compartments spatially segregate from the cytoplasm and can regulate aberrant protein aggregation, a process linked to several medical conditions, including Alzheimer's and Parkinson's diseases. Yet the mechanisms by which these droplet-like compartments affect protein aggregation remain unknown. Here, we combine kinetic theory of protein aggregation and liquid-liquid phase separation to study the spatial control of irreversible protein aggregation in the presence of liquid compartments. We find that, even for weak interactions between the compartment constituents and the aggregating monomers, aggregates are strongly enriched inside the liquid compartment relative to the surrounding cytoplasm. We show that this enrichment is caused by a positive feedback mechanism of aggregate nucleation and growth which is mediated by a flux maintaining the phase equilibrium between the compartment and the cytoplasm. Our model predicts that the compartment volume that maximizes aggregate enrichment in the compartment is determined by the reaction orders of aggregate nucleation. The underlying mechanism of aggregate enrichment could be used to confine cytotoxic protein aggregates inside droplet-like compartments suggesting potential new avenues against aberrant protein aggregation. Our findings could also represent a common mechanism for the spatial control of irreversible chemical reactions in general

Aggregation controlled by condensate rheology

Biomolecular condensates in living cells can exhibit a complex rheology, including viscoelastic and glassy behavior. This rheological behavior of condensates was suggested to regulate polymerization of cytoskeletal filaments and aggregation of amyloid fibrils. Here, we theoretically investigate how the rheological properties of condensates can control the formation of linear aggregates. To this end, we propose a kinetic theory for linear aggregation in coexisting phases, which accounts for the aggregate size distribution and the exchange of aggregates between inside and outside of condensates. The rheology of condensates is accounted in our model via aggregate mobilities that depend on aggregate size. We show that condensate rheology determines whether aggregates of all sizes or dominantly small aggregates are exchanged between condensate inside and outside on the timescale of aggregation. As a result, the ratio of aggregate numbers inside to outside of condensates differs significantly. Strikingly, we also find that weak variations in the rheological properties of condensates can lead to a switch-like change of the number of aggregates. These results suggest a possible physical mechanism for how living cells could control linear aggregation in a switch-like fashion through variations in condensate rheology

Enhanced potency of aggregation inhibitors mediated by liquid condensates

Liquid condensates are membraneless organelles that form via phase separation in living cells. These condensates provide unique heterogeneous environments that have much potential in regulating a range of biochemical processes from gene expression to filamentous protein aggregation—a process linked to Alzheimer's and Parkinson's diseases. Here we theoretically study the physical interplay between protein aggregation, its inhibition, and liquid-liquid phase separation. Our key finding is that the action of protein aggregation inhibitors can be strongly enhanced by liquid condensates. The physical mechanism of this enhancement relies on the partitioning and colocalization of inhibitors with their targets inside the liquid condensate. Our theory uncovers how the physicochemical properties of condensates can be used to modulate inhibitor potency, and we provide experimentally testable conditions under which drug potency is maximal. Our findings suggest design principles for protein aggregation inhibitors with respect to their phase-separation properties

Reaction rate theory for supramolecular kinetics: application to protein aggregation

Probing the reaction mechanisms of supramolecular processes in soft- and biological matter, such as protein aggregation, is inherently challenging. These processes emerge from the simultaneous action of multiple molecular mechanisms, each of which is associated with the rearrangement of a large number of weak bonds, resulting in a complex free energy landscape with many kinetic barriers. Reaction rate measurements of supramolecular processes at different temperatures can offer unprecedented insights into the underlying molecular mechanisms and their thermodynamic properties. However, to be able to interpret such measurements in terms of the underlying microscopic mechanisms, a key challenge is to establish which properties of the complex free energy landscapes are probed by the reaction rate. Here, we present a reaction rate theory for supramolecular kinetics based on Kramers rate theory for diffusive reactions over multiple kinetic barriers, and apply the results to protein aggregation. Using this framework and Monte Carlo simulations, we show that reaction rates for protein aggregation are of the Arrhenius-Eyring type and that the associated activation energies probe only one relevant barrier along the respective free energy landscapes. We apply this advancement to interpret, both in experiments and in coarse-grained computer simulations, reaction rate measurements of amyloid aggregation kinetics in terms of the underlying molecular mechanisms and associated thermodynamic signatures. Our results establish a general platform for probing the mechanisms and energetics of supramolecular phenomena in soft- and biological matter using the framework of chemical kinetics

Kinetic constraints on self-assembly into closed supramolecular structures

Many biological and synthetic systems exploit self-assembly to generate highly intricate closed supramolecular architectures, ranging from self-assembling cages to viral capsids. The fundamental design principles that control the structural determinants of the resulting assemblies are increasingly well-understood, but much less is known about the kinetics of such assembly phenomena and it remains a key challenge to elucidate how these systems can be engineered to assemble in an efficient manner and avoid kinetic trapping. We show here that simple scaling laws emerge from a set of kinetic equations describing the self-assembly of identical building blocks into closed supramolecular structures and that this scaling behavior provides general rules that determine efficient assembly in these systems. Using this framework, we uncover the existence of a narrow range of parameter space that supports efficient self-assembly and reveal that nature capitalizes on this behavior to direct the reliable assembly of viral capsids on biologically relevant timescales

Kinetics of spontaneous filament nucleation via oligomers: Insights from theory and simulation

Nucleation processes are at the heart of a large number of phenomena, from cloud formation to protein crystallization. A recently emerging area where nucleation is highly relevant is the initiation of filamentous protein self-assembly, a process that has broad implications in many research areas ranging from medicine to nanotechnology. As such, spontaneous nucleation of protein fibrils has received much attention in recent years with many theoretical and experimental studies focussing on the underlying physical principles. In this paper we make a step forward in this direction and explore the early time behaviour of filamentous protein growth in the context of nucleation theory. We first provide an overview of the thermodynamics and kinetics of spontaneous nucleation of protein filaments in the presence of one relevant degree of freedom, namely the cluster size. In this case, we review how key kinetic observables, such as the reaction order of spontaneous nucleation, are directly related to the physical size of the critical nucleus. We then focus on the increasingly prominent case of filament nucleation that includes a conformational conversion of the nucleating building-block as an additional slow step in the nucleation process. Using computer simulations, we study the concentration dependence of the nucleation rate. We find that, under these circumstances, the reaction order of spontaneous nucleation with respect to the free monomer does no longer relate to the overall physical size of the nucleating aggregate but rather to the portion of the aggregate that actively participates in the conformational conversion. Our results thus provide a novel interpretation of the common kinetic descriptors of protein filament formation, including the reaction order of the nucleation step or the scaling exponent of lag times, and put into perspective current theoretical descriptions of protein aggregation.We acknowledge support from the Human Frontier Science Program and Emmanuel College (A.Š.), St John’s and Peterhouse Colleges (T.C.T.M.), the Swiss National Science Foundation (T.C.T.M.), the Biotechnology and Biological Sciences Research Council (T.P.J.K.), the Frances and Augustus Newman Foundation (T.P.J.K.), the European Research Council (T.C.T.M., T.P.J.K., and D.F.), and the Engineering and Physical Sciences Research Council (D.F.)

Small-molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer's disease.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes