RESUMO
Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol.
Assuntos
alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Concentração de Íons de Hidrogênio , Humanos , Multimerização Proteica , Agregados Proteicos , Concentração Osmolar , Doença de Parkinson/metabolismoRESUMO
Functional amyloids formed by the protein FapC in Pseudomonas bacteria are key structural components of Pseudomonas biofilms, which mediate chronic infections and also contribute to antimicrobial resistance. Here, we combine kinetic experiments with mechanistic modelling to probe the role of surfaces in FapC functional amyloid formation. We find that nucleation of new fibrils is predominantly heterogeneous in vitro, being catalysed by reaction vessel walls but not by the air/water interface. Removal of such interfaces by using microdroplets greatly slows heterogeneous nucleation and reveals a hitherto undetected fibril surface-catalysed "secondary nucleation" reaction step. We tune the degree of catalysis by varying the interface chemistry of the reaction vessel and by adding nanoparticles with tailored surface properties that catalyse fibril nucleation. In so doing, we discover that the rate of nucleation is controlled predominantly by the strength with which FapC binds to the catalytic sites on the interface, and by its surface area. Surprisingly, neither primary nucleation rate nor catalytic site binding strength appear closely correlated to the charge and hydrophilicity of the interface. This indicates the importance of considering experimental design in terms of surface chemistry of the reaction container while also highlighting the notion that fibril nucleation during protein aggregation is a heterogeneous process.
Assuntos
Amiloide , Propriedades de Superfície , Amiloide/química , Amiloide/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cinética , Biofilmes , Pseudomonas/metabolismo , Nanopartículas/química , Interações Hidrofóbicas e HidrofílicasRESUMO
Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.
Assuntos
Amiloide , Amiloide/metabolismo , Amiloide/química , Cinética , Humanos , Resistência ao Cisalhamento , Agregados Proteicos , Peptídeos/química , Peptídeos/metabolismo , Doença de Alzheimer/metabolismoRESUMO
Protein aggregation is a key process in the development of many neurodegenerative disorders, including dementias such as Alzheimer's disease. Significant progress has been made in understanding the molecular mechanisms of aggregate formation in pure buffer systems, much of which was enabled by the development of integrated rate laws that allowed for mechanistic analysis of aggregation kinetics. However, in order to translate these findings into disease-relevant conclusions and to make predictions about the effect of potential alterations to the aggregation reactions by the addition of putative inhibitors, the current models need to be extended to account for the altered situation encountered in living systems. In particular, in vivo, the total protein concentrations typically do not remain constant and aggregation-prone monomers are constantly being produced but also degraded by cells. Here, we build a theoretical model that explicitly takes into account monomer production, derive integrated rate laws and discuss the resulting scaling laws and limiting behaviours. We demonstrate that our models are suited for the aggregation-prone Huntington's disease-associated peptide HttQ45 utilizing a system for continuous in situ monomer production and the aggregation of the tumour suppressor protein P53. The aggregation-prone HttQ45 monomer was produced through enzymatic cleavage of a larger construct in which a fused protein domain served as an internal inhibitor. For P53, only the unfolded monomers form aggregates, making the unfolding a rate-limiting step which constitutes a source of aggregation-prone monomers. The new model opens up possibilities for a quantitative description of aggregation in living systems, allowing for example the modelling of inhibitors of aggregation in a dynamic environment of continuous protein synthesis.
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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.
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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.
Assuntos
Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Amiloide/metabolismo , Amiloide/química , Doença de Alzheimer/metabolismo , CinéticaRESUMO
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.
Assuntos
Entropia , Proteólise , Proteoma , Proteoma/metabolismo , Humanos , Animais , Teorema de Bayes , Proteostase , Cinética , AMP Cíclico/metabolismo , Serina-Treonina Quinases TOR/metabolismo , GMP Cíclico/metabolismoRESUMO
The self-assembly of the amyloid ß 42 (Aß42) peptide is linked to Alzheimer's disease, and oligomeric intermediates are linked to neuronal cell death during the pathology of the disease. These oligomers are produced prolifically during secondary nucleation, by which the aggregation of monomers is catalyzed on fibril surfaces. Significant progress has been made in understanding the aggregation mechanism of Aß42; still, a detailed molecular-level understanding of secondary nucleation is lacking. Here, we explore the role of four hydrophobic residues on the unstructured N-terminal region of Aß42 in secondary nucleation. We create eight mutants with single substitutions at one of the four positionsâAla2, Phe4, Tyr10, and Val12âto decrease the hydrophobicity at respective positions (A2T, A2S, F4A, F4S, Y10A, Y10S, V12A, and V12S) and one mutant (Y10F) to remove the polar nature of Tyr10. Kinetic analyses of aggregation data reveal that the hydrophobicity at the N-terminal region of Aß42, especially at positions 10 and 12, affects the rate of fibril mass generated via secondary nucleation. Cryo-electron micrographs reveal that most of the mutants with lower hydrophobicity form fibrils that are markedly longer than WT Aß42, in line with the reduced secondary nucleation rates for these peptides. The dominance of secondary nucleation, however, is still retained in the aggregation mechanism of these mutants because the rate of primary nucleation is even more reduced. This highlights that secondary nucleation is a general phenomenon that is not dependent on any one particular feature of the peptide and is rather robust to sequence perturbations.
Assuntos
Peptídeos beta-Amiloides , Peptídeos beta-Amiloides/genéticaRESUMO
Protein self-assembly into amyloid fibrils underlies several neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that the small oligomers formed during this process constitute neurotoxic molecular species associated with amyloid aggregation. Targeting the formation of oligomers represents, therefore, a possible therapeutic avenue to combat these diseases. However, it remains challenging to establish which microscopic steps should be targeted to suppress most effectively the generation of oligomeric aggregates. Recently, we have developed a kinetic model of oligomer dynamics during amyloid aggregation. Here, we use this approach to derive explicit scaling relationships that reveal how key features of the time evolution of oligomers, including oligomer peak concentration and lifetime, are controlled by the different rate parameters. We discuss the therapeutic implications of our framework by predicting changes in oligomer concentrations when the rates of the individual microscopic events are varied. Our results identify the kinetic parameters that control most effectively the generation of oligomers, thus opening a new path for the systematic rational design of therapeutic strategies against amyloid-related diseases.
Assuntos
Amiloide , Doenças Neurodegenerativas , Amiloide/metabolismo , Proteínas Amiloidogênicas , Humanos , CinéticaRESUMO
The pathology of Alzheimer's disease is connected to the aggregation of ß-amyloid (Aß) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, Aß37 and Aß38, as pure peptides and in mixtures with Aß40 and Aß42. A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform Aß42. Through kinetic analysis, we show that both Aß37 and Aß38 self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar ß-sheet-rich aggregates, albeit on a longer timescale than Aß40 or Aß42. Additionally, we show that the shorter alloforms co-aggregate with Aß40, affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither Aß37 nor Aß38 forms co-aggregates with Aß42; however, both short alloforms reduce the rate of Aß42 aggregation in a concentration-dependent manner. Finally, we show that the aggregation of Aß42 is more significantly impeded by a combination of Aß37, Aß38, and Aß40 than by any of these alloforms independently. These results demonstrate that the aggregation of any given Aß alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain.
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The self-assembly of peptides and proteins into amyloid fibrils plays a causative role in a wide range of increasingly common and currently incurable diseases. The molecular mechanisms underlying this process have recently been discovered, prompting the development of drugs that inhibit specific reaction steps as possible treatments for some of these disorders. A crucial part of treatment design is to determine how much drug to give and when to give it, informed by its efficacy and intrinsic toxicity. Since amyloid formation does not proceed at the same pace in different individuals, it is also important that treatment design is informed by local measurements of the extent of protein aggregation. Here, we use stochastic optimal control theory to determine treatment regimens for inhibitory drugs targeting several key reaction steps in protein aggregation, explicitly taking into account variability in the reaction kinetics. We demonstrate how these regimens may be updated "on the fly" as new measurements of the protein aggregate concentration become available, in principle, enabling treatments to be tailored to the individual. We find that treatment timing, duration, and drug dosage all depend strongly on the particular reaction step being targeted. Moreover, for some kinds of inhibitory drugs, the optimal regimen exhibits high sensitivity to stochastic fluctuations. Feedback controls tailored to the individual may therefore substantially increase the effectiveness of future treatments.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Retroalimentação , Humanos , Cinética , Agregados Proteicos/efeitos dos fármacos , Processos EstocásticosRESUMO
Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation. We use this approach to investigate the aggregation of α-synuclein (αS), a synaptic protein implicated in Parkinson's disease and find a large population of oligomeric species. Our results show that these are primary oligomers, formed directly from monomeric species, rather than oligomers formed by secondary nucleation processes, and that they are short-lived, the majority of them dissociates rather than converts to fibrils. As demonstrated here, EPR offers the means to detect such short-lived intermediate species directly in situ. As it relies only on the change in size of the detected species, it will be applicable to a wide range of self-assembling systems, making accessible the kinetics of intermediates and thus allowing the determination of their rates of formation and conversion, key processes in the self-assembly reaction.
Assuntos
Agregados Proteicos , Multimerização Proteica , alfa-Sinucleína/química , Cinética , Estrutura Quaternária de ProteínaRESUMO
Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.
Assuntos
Hidrogéis , Peptídeos , CinéticaRESUMO
The misfolding and aberrant aggregation of proteins into fibrillar structures is a key factor in some of the most prevalent human diseases, including diabetes and dementia. Low molecular weight oligomers are thought to be a central factor in the pathology of these diseases, as well as critical intermediates in the fibril formation process, and as such have received much recent attention. Moreover, on-pathway oligomeric intermediates are potential targets for therapeutic strategies aimed at interrupting the fibril formation process. However, a consistent framework for distinguishing on-pathway from off-pathway oligomers has hitherto been lacking and, in particular, no consensus definition of on- and off-pathway oligomers is available. In this paper, we argue that a non-binary definition of oligomers' contribution to fibril-forming pathways may be more informative and we suggest a quantitative framework, in which each oligomeric species is assigned a value between 0 and 1 describing its relative contribution to the formation of fibrils. First, we clarify the distinction between oligomers and fibrils, and then we use the formalism of reaction networks to develop a general definition for on-pathway oligomers, that yields meaningful classifications in the context of amyloid formation. By applying these concepts to Monte Carlo simulations of a minimal aggregating system, and by revisiting several previous studies of amyloid oligomers in light of our new framework, we demonstrate how to perform these classifications in practice. For each oligomeric species we obtain the degree to which it is on-pathway, highlighting the most effective pharmaceutical targets for the inhibition of amyloid fibril formation.
RESUMO
The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer's disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aß42 peptide implicated in Alzheimer's disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/toxicidade , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Cinética , Bicamadas Lipídicas , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Imagem Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/toxicidadeRESUMO
The aggregation of peptides and proteins into amyloid fibrils is a molecular self-assembly phenomenon associated with both biological function and malfunction, notably in the context of neurodegenerative diseases. Oligomeric species formed early in the aggregation process are generally associated with cytotoxicity. Extrinsic molecules such as peptides have been found to influence amyloid formation kinetics and regulate this cellular process. Here, we use single-molecule FRET and bulk assays combined with global kinetic analysis to study quantitatively the effect of an 8-residue peptide (LQVNIGNR) on fibril formation by the yeast prion protein Ure2. This peptide, which is derived from a segment of the Ure2 prion domain, forms vesicular assemblies that accelerate fibril formation of Ure2 by promoting conformational conversion of oligomeric intermediates into fibrillar species in a catalytic manner. This reduces oligomer longevity and consequently ameliorates cytotoxicity. The LQVNIGNR peptide was found to accelerate fibril formation of unrelated proteins including Tau and α-Synuclein, suggesting a general ability to catalyse fibrillation. This study provides a general strategy for investigating the microscopic mechanism of extrinsic factors on amyloid aggregation. This approach can readily be applied to other amyloid systems and demonstrates that acceleration of oligomer conversion is a promising strategy to reduce amyloid toxicity.
Assuntos
Amiloide , Príons , Catálise , Cinética , alfa-SinucleínaRESUMO
The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer's and Parkinson's diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cinética , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Amiloidose , Modelos Teóricos , Agregados Proteicos , TermodinâmicaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Oligomeric species populated during the aggregation of the Aß42 peptide have been identified as potent cytotoxins linked to Alzheimer's disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aß42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aß42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Simulação por Computador , Humanos , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Multimerização ProteicaRESUMO
The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aß40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.