Nucleation-dependent Polymerization Mechanism Research Articles

Rationalizing the Role of Monosodium Glutamate in the Protein Aggregation Through Biophysical Approaches: Potential Impact on Neurodegeneration.

Monosodium glutamate (MSG) is the world’s most extensively used food additive and is generally recognized as safe according to the FDA. However, it is well reported that MSG is associated with a number of neurological diseases, and in turn, neurological diseases are associated with protein aggregation. This study rationalized the role of MSG in protein aggregation using different biophysical techniques such as absorption, far-UV CD, DLS, and ITC. Kinetic measurements revealed that MSG causes significant enhancement of aggregation of BSA through a nucleation-dependent polymerization mechanism. Also, CTAB-BSA aggregation is enhanced by MSG significantly. MSG-induced BSA aggregation also exhibits the formation of irreversible aggregates, temperature dependence, non-Arrhenius behavior, and enhancement of hydrodynamic diameter. From the isothermal titration calorimetry measurement, the significant endothermic heat of the interaction of BSA-MSG indicates that protein aggregation may be due to the coupling of MSG with the protein. The determined enthalpy change (ΔH) is largely positive, also suggesting an endothermic nature, whereas entropy change (ΔS) is positive and Gibbs free energy change (ΔG) is largely negative, suggesting the spontaneous nature of the interaction. Furthermore, even a low concentration of MSG is involved in the unfolding of the secondary structure of protein with the disappearance of original peaks and the formation of a unique peak in the far-UV CD, which is an attention-grabbing observation. This is the first investigation which links the dietary MSG with protein aggregation and thus will be very instrumental in understanding the mechanism of various MSG-related human physiological as well as neurological diseases.

Recent progress on understanding the mechanisms of amyloid nucleation.

Amyloid fibrils are supramolecular protein assemblies with a fibrous morphology and cross-β structure. The formation of amyloid fibrils typically follows a nucleation-dependent polymerization mechanism, in which a one-step nucleation scheme has widely been accepted. However, a variety of oligomers have been identified in early stages of fibrillation, and a nucleated conformational conversion (NCC) mechanism, in which oligomers serve as a precursor of amyloid nucleation and convert to amyloid nuclei, has been proposed. This development has raised the need to consider more complicated multi-step nucleation processes in addition to the simplest one-step process, and evidence for the direct involvement of oligomers as nucleation precursors has been obtained both experimentally and theoretically. Interestingly, the NCC mechanism has some analogy with the two-step nucleation mechanism proposed for inorganic and organic crystals and protein crystals, although a more dramatic conformational conversion of proteins should be considered in amyloid nucleation. Clarifying the properties of the nucleation precursors of amyloid fibrils in detail, in comparison with those of crystals, will allow a better understanding of the nucleation of amyloid fibrils and pave the way to develop techniques to regulate it.

Inferring Mechanistic Parameters from Amyloid Formation Kinetics by Approximate Bayesian Computation

Amyloid formation is implicated in a number of human diseases, and is thought to proceed via a nucleation-dependent polymerization mechanism. Experimenters often wish to relate changes in amyloid formation kinetics, for example, in response to small molecules to specific mechanistic steps along this pathway. However, fitting kinetic fibril formation data to a complex model including explicit rate constants results in an ill-posed problem with a vast number of potential solutions. The levels of uncertainty remaining in parameters calculated from these models, arising both from experimental noise and high levels of degeneracy or codependency in parameters, is often unclear. Here, we demonstrate that a combination of explicit mathematical models with an approximate Bayesian computation approach can be used to assign the mechanistic effects of modulators on amyloid fibril formation. We show that even when exact rate constants cannot be extracted, parameters derived from these rate constants can be recovered and used to assign mechanistic effects and their relative magnitudes with a great deal of confidence. Furthermore, approximate Bayesian computation provides a robust method for visualizing uncertainty remaining in the model parameters, regardless of its origin. We apply these methods to the problem of heparin-mediated tau polymerization, which displays complex kinetic behavior not amenable to analysis by more traditional methods. Our analysis indicates that the role of heparin cannot be explained by enhancement of nucleation alone, as has been previously proposed. The methods described here are applicable to a wide range of systems, as models can be easily adapted to account for new reactions and reversibility.

Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation

Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40 and 50% TFE to proceed α to β transition, suggesting the formation of an intermolecular β-structure. AFM images confirmed the nature of Fib aggregates at 40 and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular β-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intramolecular hydrogen bonds over intermolecular hydrogen bonds leading to helix formation. To conclude, protein aggregation is accompanied by the formation of β-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the β-sheet and α-helix conformation.

Ill-Posed? Not a Problem: Inferring Mechanisms of Action from Amyloid Formation Kinetics using Approximate Bayesian Computation

Amyloid formation is implicated in a number of human diseases, and is thought to proceed via a nucleation-dependent polymerization mechanism wherein small concentrations of “nuclei” form initially, followed by the rapid, highly favorable addition of further monomer to nuclei/fibril ends. This type of behavior results in highly cooperative, sigmoidal fibrillization kinetics. In many cases, experimenters wish to relate changes in the observed kinetics, for example in response to small molecule effectors, to specific mechanistic steps along this pathway. However, fitting kinetic fibril formation data to a complex model including explicit rate constants results in an ill-posed problem, with a vast number of potential solutions. The amount of uncertainty remaining in parameters calculated from these models, which arises both from experimental noise and high levels of covariance in parameters, is often unclear.Here, we demonstrate the utility of combining these explicit mathematical models with approximate Bayesian computational (ABC) techniques to assign mechanistic roles to small molecule effectors of amyloid fibril formation. We show even when exact rate constants cannot be extracted from bulk amyloid formation kinetic data, the ratios and products of these rate constants can be recovered and used to assign mechanistic effects and their relative magnitudes with confidence. Furthermore, ABC provides a robust method for visualizing uncertainty remaining in the model parameters, regardless of its origin. We apply these methods to the problem of heparin-mediated tau polymerization, which displays complex kinetic behavior not amenable to analysis by more traditional methods. Our studies indicate that heparin's effect cannot be explained by enhancement of nucleation alone, as has been previously proposed. These methods are applicable to a wide range of systems, as models can be easily adapted to account for new reactions and reversibility.

Amyloid Fibril Formation by the Chain B Subunit of Monellin Occurs by a Nucleation-Dependent Polymerization Mechanism

Proteins possessing very different structures, or even no structure, form amyloid fibrils that are very similar in internal structure. This suggests that the mechanisms by which amyloid fibrils form might be very similar, irrespective of whether the fibrils are associated with disease or with normal cellular function, or even if they have no physiological importance. In this context, it is important to have a model protein system whose amyloid fibril formation is robust in its reproducibility, which can reveal the fundamentals of the amyloid fibril reaction that may be applicable to all proteins. In this study, the aggregation mechanism of amyloid fibril formation by chain B of the heterodimeric protein monellin has been elucidated in detail. It is shown that the aggregation reaction meets all the stringent kinetic criteria of a homogeneous nucleation-dependent polymerization mechanism, which is valid over a wide range of protein concentrations. Quantitative analyses of the kinetic data using one approach based on features of the entire kinetic curve, and another based on only the initial rate of aggregation, indicate that the thermodynamic nucleus is a dimer. Spherical oligomers are observed by atomic force microscopy to form transiently early during fibril formation but are off-pathway to the direct fibril formation pathway. It is shown that amyloid fibril formation can be prevented by the addition of chain A of monellin at early stages of chain B aggregation: the two free chains combine to form native monellin, which leads to the dissociation of early aggregates.

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20 μM). Importantly, when the 5 μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.

Mechanisms of Ataxin-3 Misfolding and Fibril Formation: Kinetic Analysis of a Disease-associated Polyglutamine Protein

The polyglutamine diseases are a family of nine proteins where intracellular protein misfolding and amyloid-like fibril formation are intrinsically coupled to disease. Previously, we identified a complex two-step mechanism of fibril formation of pathologically expanded ataxin-3, the causative protein of spinocerebellar ataxia type-3 (Machado-Joseph disease). Strikingly, ataxin-3 lacking a polyglutamine tract also formed fibrils, although this occurred only via a single-step that was homologous to the first step of expanded ataxin-3 fibril formation. Here, we present the first kinetic analysis of a disease-associated polyglutamine repeat protein. We show that ataxin-3 forms amyloid-like fibrils by a nucleation-dependent polymerization mechanism. We kinetically model the nucleating event in ataxin-3 fibrillogenesis to the formation of a monomeric thermodynamic nucleus. Fibril elongation then proceeds by a mechanism of monomer addition. The presence of an expanded polyglutamine tract leads subsequently to rapid inter-fibril association and formation of large, highly stable amyloid-like fibrils. These results enhance our general understanding of polyglutamine fibrillogenesis and highlights the role of non-poly(Q) domains in modulating the kinetics of misfolding in this family.

Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties

Pancreatic amyloid is found in more than 95 % of type II diabetes patients. Pancreatic amyloid is formed by the aggregation of islet amyloid polypeptide (hIAPP or amylin), which is a 37-residue peptide. Because pancreatic amyloid is cytotoxic, it is believed that its formation is directly associated with the development of the disease. We recently showed that hIAPP amyloid formation follows the nucleation-dependent polymerization mechanism and proceeds via a conformational transition of soluble hIAPP into aggregated β-sheets. Here, we report that the penta- and hexapeptide sequences, hIAPP(23–27) (FGAIL) and hIAPP(22–27) (NFGAIL) of hIAPP are sufficient for the formation of β-sheet-containing amyloid fibrils. Although these two peptides differ by only one amino acid residue, they aggregate into completely different fibrillar assemblies. hIAPP(23–27) (FGAIL) fibrils self-assemble laterally into unusually broad ribbons, whereas hIAPP(22–27) (NFGAIL) fibrils coil around each other in a typical amyloid fibril morphology. hIAPP(20–27) (SNNFGAIL) also aggregates into β-sheet-containing fibrils, whereas no amyloidogenicity is found for hIAPP(24–27) (GAIL), indicating that hIAPP(23–27) (FGAIL) is the shortest fibrillogenic sequence of hIAPP. Insoluble amyloid formation by the partial hIAPP sequences followed kinetics that were consistent with a nucleation-dependent polymerization mechanism. hIAPP(22–27) (NFGAIL), hIAPP(20–27) (SNNFGAIL), and also the known fibrillogenic sequence, hIAPP(20–29) (SNNFGAILSS) exhibited significantly lower kinetic and thermodynamic solubilities than the pentapeptide hIAPP(23–27) (FGAIL). Fibrils formed by all short peptide sequences and also by hIAPP(20–29) were cytotoxic towards the pancreatic cell line RIN5fm, whereas no cytotoxicity was observed for the soluble form of the peptides, a notion that is consistent with hIAPP cytotoxicity. Our results suggest that a penta- and hexapeptide sequence of an appropriate amino acid composition can be sufficient for β-sheet and amyloid fibril formation and cytotoxicity and may assist in the rational design of inhibitors of pancreatic amyloid formation or other amyloidosis-related diseases.

Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in Vitro

Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer’s disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into β-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 μM peptide, we found that this state was mostly populated at about 45 °C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 °C. The state at 45 °C was also found to be populated at 4.25 M GdnHCl at 25 °C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of β-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases.

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease?

NAC is a 35-amino-acid peptide which has been isolated from the insoluble core of Alzheimer's disease (AD) amyloid plaque. It is a fragment of alpha-synuclein (or NACP), a neuronal protein of unknown function. We noted a striking sequence similarity between NAC, the carboxyl terminus of the beta-amyloid protein, and a region of the scrapie prion protein (PrP) which has been implicated in amyloid formation. NAC was prepared by chemical synthesis and was found to form amyloid fibrils via a nucleation-dependent polymerization mechanism. NAC amyloid fibrils effectively seed beta 1-40 amyloid formation. Amyloid fibrils comprising peptide models of the homologous beta and PrP sequences were also found to seed amyloid formation by NAC. The in vitro model studies presented here suggest that seeding of NAC amyloid formation by the beta-amyloid protein, or seeding of amyloid fibrils of the beta-amyloid protein by NAC, may occur in vivo. Accumulation of ordered NAC aggregates in the synapse may be responsible for the neurodegeneration observed in AD and the prion disorders. Alternatively, neurodegeneration may be caused by the loss of alpha-synuclein (NACP) function.

Predisposition of prion protein homozygotes to Creutzfeldt-Jakob disease can be explained by a nucleation-dependent polymerization mechanism

Predisposition of prion protein homozygotes to Creutzfeldt-Jakob disease can be explained by a nucleation-dependent polymerization mechanism