Fitting yeast and mammalian prion aggregation kinetic data with the Finke-Watzky two-step model of nucleation and autocatalytic growth

Recently, we reported 14 amyloid protein aggregation kinetic data sets that were fit using the "Ockham's razor"/minimalistic Finke-Watzky (F-W) two-step model of slow nucleation (A --> B, rate constant k 1) and fast autocatalytic growth (A + B --> 2B, rate constant k 2), yielding quantitative (average) rate constants for nucleation ( k 1) and growth ( k 2), where A is the monomeric protein and B is the polymeric protein [Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427]. Herein, we apply the F-W model to 27 representative prion aggregation kinetic data sets obtained from the literature. Each prion data set was successfully fit with the F-W model, including three different yeast prion proteins (Sup35p, Ure2p, and Rnq1p) as well as mouse and human prions. These fits yield the first quantitative rate constants for the steps of nucleation and growth in prion aggregation. Examination of a Sup35p system shows that the same rate constants are obtained for nucleation and for growth within experimental error, regardless of which of six physical methods was used, a unique set of important control experiments in the protein aggregation literature. Also provided herein are analyses of several factors influencing the aggregation of prions such as glutamine/asparagine rich regions and the number of oligopeptide repeats in the prion domain. Where possible, verification or refutation of previous correlations to glutamine/asparagine regions, or the number of repeat sequences, in literature aggregation kinetics is given in light of the quantitative rate constants obtained herein for nucleation and growth during prion aggregation. The F-W model is then contrasted to four literature mechanisms that address the molecular picture of prion transmission and propagation. Key limitations of the F-W model are listed to prevent overinterpretation of the data being analyzed, limitations that derive ultimately from the model's simplicity. Finally, possible avenues of future research are suggested.     

Evidence for the existence of a secondary pathway for fibril growth during the aggregation of tau

The mechanism of amyloid fibril formation by proteins has been classically described by the nucleation-dependent polymerization (NDP) model, which makes certain predictions regarding the kinetics of fibrillation. All proteins whose aggregation conforms to the NDP model display a t(2) time dependence for their initial reaction profile. However, there are proteins whose aggregation reactions have kinetic signatures of a flat lag phase followed by an exponential rise in fibril mass, which does not conform to the NDP model. Amyloid fibril formation by tau, a microtubule-associated protein whose aggregation to form neurofibrillary tangles is implicated in Alzheimer's disease and other tauopathies, in the presence of inducers such as heparin and fatty acid micelles, has always been traditionally described by a ligand-induced NDP model. In this study, the existence of a secondary pathway for fibril growth during the aggregation of the functional, repeat domain of tau in the presence of heparin has been established. Both kinetic and accessory evidence are provided for the existence of this pathway, which is shown to augment the primary homogeneous nucleation pathway. From the kinetic data, the main secondary pathway that is operative appears to be fibril fragmentation but other pathways such as branching or secondary nucleation may also be operative.     

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.     

Non-native aggregation of alpha-chymotrypsinogen occurs through nucleation and growth with competing nucleus sizes and negative activation energies

The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.     

α-Synuclein aggregation variable temperature and variable pH kinetic data: A re-analysis using the Finke–Watzky 2-step model of nucleation and autocatalytic growth

The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k₁) and autocatalytic growth (A + B → 2B, rate constant k₂). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k₁) and growth (k₂) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG^‡, ΔH^‡, and ΔS^‡ are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔG^‡_nucleation = 23(3) kcal/mol; ΔG^‡_growth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k₁ value at pH ~ 3, as well as minimal k₁ near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k₂ values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k₁ vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k₁, nucleation step of α-synuclein. The chemically well-defined nucleation (k₁) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k₂ × [A]₀ (where k₂ is the rate constant for autocatalytic growth and [A]₀ is the initial protein concentration) is related to the empirical constant, k_app obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.     

Kinetically competing huntingtin aggregation pathways control amyloid polymorphism and properties

In polyglutamine (polyQ) containing fragments of the Huntington's disease protein huntingtin (htt), the N-terminal 17 amino acid htt(NT) segment serves as the core of α-helical oligomers whose reversible assembly locally concentrates the polyQ segments, thereby facilitating polyQ amyloid nucleation. A variety of aggregation inhibitors have been described that achieve their effects by neutralizing this concentrating function of the htt(NT) segment. In this paper we characterize the nature and limits of this inhibition for three means of suppressing htt(NT)-mediated aggregation. We show that the previously described action of htt(NT) peptide-based inhibitors is solely due to their ability to suppress the htt(NT)-mediated aggregation pathway. That is, under htt(NT) inhibition, nucleation of polyQ amyloid formation by a previously described alternative nucleation mechanism proceeds unabated and transiently dominates the aggregation process. Removal of the bulk of the htt(NT) segment by proteolysis or mutagenesis also blocks the htt(NT)-mediated pathway, allowing the alternative nucleation pathway to dominate. In contrast, the previously described immunoglobulin-based inhibitor, the antihtt(NT) V(L) 12.3 protein, effectively blocks both amyloid pathways, leading to stable accumulation of nonamyloid oligomers. These data show that the htt(NT)-dependent and -independent pathways of amyloid nucleation in polyQ-containing htt fragments are in direct kinetic competition. The results illustrate how amyloid polymorphism depends on assembly mechanism and kinetics and have implications for how the intracellular environment can influence aggregation pathways.     

A kinetic model for binding protein-mediated arabinose transport.

A kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein-dependent transport. The transport phenotypes of the 18 variant arabinose-binding proteins analyzed by Kehres and Hogg (1992, Protein Sci. 1, 1652-1660) (wild type and 17 mutants) are interpreted to mean that in wild-type arabinose uptake the forward transport rate (k(for)) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that k(for) dominance is preserved in all of the binding protein surface mutants. The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems.     

Model Discrimination and Mechanistic Interpretation of Kinetic Data in Protein Aggregation Studies

Given the importance of protein aggregation in amyloid diseases and in the manufacture of protein pharmaceuticals, there has been increased interest in measuring and modeling the kinetics of protein aggregation. Several groups have analyzed aggregation data quantitatively, typically measuring aggregation kinetics by following the loss of protein monomer over time and invoking a nucleated growth mechanism. Such analysis has led to mechanistic conclusions about the size and nature of the nucleus, the aggregation pathway, and/or the physicochemical properties of aggregation-prone proteins. We have examined some of the difficulties that arise when extracting mechanistic meaning from monomer-loss kinetic data. Using literature data on the aggregation of polyglutamine, a mutant β-clam protein, and protein L, we determined parameter values for 18 different kinetic models. We developed a statistical model discrimination method to analyze protein aggregation data in light of competing mechanisms; a key feature of the method is that it penalizes overparameterization. We show that, for typical monomer-loss kinetic data, multiple models provide equivalent fits, making mechanistic determination impossible. We also define the type and quality of experimental data needed to make more definitive conclusions about the mechanism of aggregation. Specifically, we demonstrate how direct measurement of fibril size provides robust discrimination.     

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.     

Time scale of protein aggregation dictated by liquid-liquid demixing

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington's disease

ABSTRACT: BACKGROUND: Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington's disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the lengthdependent nucleation and the nucleation-dependent elongation. In contrast to the repeatlength dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process. RESULTS: Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression. CONCLUSIONS: Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.     

Absolute correlation between lag time and growth rate in the spontaneous formation of several amyloid-like aggregates and fibrils

The formation of polypeptide aggregates, including amyloid fibrils and prions, is a biochemical process of considerable interest in the context of its association with ageing and neurodegeneration. Aggregation occurs typically with a lag phase and a growth phase that reflect an underlying nucleation-polymerisation mechanism. While the propensity of nucleation can be estimated from the lag time t(l), the efficiency of growth is represented by the growth rate k(g). Here, I have analysed the absolute k(g) and t(l) values from a total of 298 samples prepared from insulin, glucagon and different sequence variants of the Alzheimer's Abeta(1-40) peptide. Although these samples differ in the conditions of aggregation, systematic comparison reveals an overall similarity in the plot of k(g)versus t(l). The plot fits readily with the simple equation k(g)=alpha/t(l) and by using a proportionality factor alpha of 4.5. In contrast to the individual values of k(g) and t(l) that depend substantially on sequential and environmental parameters, alpha seems much less affected by such factors. These data suggest mechanistic similarities in the nucleation behaviour of different amyloid-like fibrils and aggregates.     

NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of alpha-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of alpha-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with alpha-synuclein by NMR and assigned the binding site to C-terminal residues 109-140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 --> +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by approximately 10(4) and the rate of monomer addition approximately 40-fold. Significant secondary structure is not induced in monomeric alpha-synuclein by polyamines at 15 degrees C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the beta-sheet conformation characteristic of fibrillar alpha-synuclein. We conclude that the C-terminal domain acts as a regulator of alpha-synuclein aggregation.     

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Water Molecular System Dynamics Associated with Amyloidogenic Nucleation as Revealed by Real Time Near Infrared Spectroscopy and Aquaphotomics

The formation of amyloid fibrils proceeds via a nucleation-dependent mechanism in which nucleation phase is generally associated with a high free energy resulting in the rate-limiting step. On the basis of this kinetic feature, the nucleation is one of the most crucial phases controlling the pathogenesis of amyloidoses, but little is known about the details of how protein molecules and surrounding environment vary at this stage. Here, we applied near infrared (NIR) spectral monitoring of water structural changes in real time during the nucleation-dependent fibrillation of insulin. Whilst multivariate spectral analysis in the 2050–2350 nm spectral region indicated cross-β formation, characteristic transformations of water structure have been detected in the spectral region 1300–1600 nm corresponding to the first overtone of water OH stretching vibrations. Furthermore, specific water spectral patterns (aquagrams) related to different water molecular conformations have been found along the course of protein nucleation and aggregation. Right in the beginning, dissociation of hydrogen-bonded network in bulk water and coinstantaneous protein and ion hydration were observed, followed by water hydrogen-bonded networks development, presumably forcing the nucleation. These specific transformations of water spectral pattern could be used further as a biomarker for early non-invasive diagnosis of amyloidoses prior to explosive amplification and deposits of amyloid fibrils.     

Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.     

Early events in insulin fibrillization studied by time-lapse atomic force microscopy

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 3. Stopped-flow studies with histrionicotoxin.

Rapid kinetic studies of histrionicotoxin interactions with membrane-bound acetylcholine-receptor showed a conformational change in the receptor-histironicotoxin complex as reflected by a decrease in fluorescence intensity of the extrinsic probe ethidium. The simplest kinetic mechanism consistent with the observed data is one in which a rapid preequiliibrium exists between receptor and toxin (K = 3.33 micrometers), followed by a slow conformational change (k1 congruent to 2 X 10(-2) s-1 and k-1 congruent to 1.5 X 10(-3) s-1). The overall equilibrium constant (Kov) determined from a fit of the amplitude dependence on toxin concentration had a value of 0.25 micrometer. The data preclude kinetic mechanisms where histrionicotoxin acts as an effector, shifting equilibria between preexisting, discrete, and slowly interconverting receptor forms.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington’s disease

Background

Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington??s disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the length-dependent nucleation and the nucleation-dependent elongation. In contrast to the repeat-length dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process.

Results

Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression.

Conclusions

Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.