Nucleation-dependent Kinetics Research Articles

Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

Amyloid fibrils are filamentous protein aggregates associated with the pathogenesis of a wide variety of human diseases. The formation of such aggregates typically follows nucleation-dependent kinetics, wherein the assembly and structural conversion of amyloidogenic proteins into oligomeric aggregates (nuclei) is the rate-limiting step of the overall reaction. In this study, we sought to gain structural insights into the oligomeric nuclei of the human prion protein (PrP) by preparing a series of deletion mutants lacking 14-44 of the C-terminal 107 residues of PrP and examined the kinetics and thermodynamics of these mutants in amyloid formation. An analysis of the experimental data using the concepts of the Φ-value analysis indicated that the helix 2 region (residues 168-196) acquires an amyloid-like β-sheet during nucleation, whereas the other regions preserves a relatively disordered structure in the nuclei. This finding suggests that the helix 2 region serves as the nucleation site for the assembly of amyloid fibrils.-Honda, R., Kuwata, K. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

Detergent-induced aggregation of an amyloidogenic intrinsically disordered protein

Abstract Intrinsically disordered proteins (IDPs) belong to an important class of proteins that do not fold up spontaneously. The conformational flexibility of IDPs allows them to adopt a wide range of conformations depending upon their biochemical environment. Many IDPs undergo profound conformational conversion that is often coupled to amyloid aggregation in the presence of negatively charged lipid membranes. Here, we show the effect of a well-known anionic lipid mimetic, sodium dodecyl sulfate (SDS), on the aggregation mechanism of a model amyloidogenic IDP, namely, bovine \(\upkappa \)-casein. In the absence of SDS, the aggregation kinetics of reduced and carboxymethylated (RCM) \(\upkappa \)-casein followed a nucleation dependent polymerization model that comprises both lag- and assembly phases. On the contrary, in the presence of sub-micellar concentration of SDS, the aggregation kinetics did not exhibit a lag phase and appears to follow a non-nucleation pathway. Additionally, the morphologies of the aggregates formed in the absence and presence of SDS were found to be different. In the absence of SDS, \(\upkappa \)-casein aggregation proceeded to typical amyloid fibrils, whereas, in the presence of SDS, the aggregation yielded large oligomers. Our results provide important molecular insights into the aggregation mechanism that can be utilized for the designing of novel protein/amyloid based nanomaterials with desired properties. Graphical Abstract Synopsis: An amyloidogenic intrinsically disordered protein, \(\upkappa \)-casein aggregates into amyloid fibrils via a typical nucleation-dependent kinetics that exhibits a lag-phase. However, in the presence of an anionic surfactant, such as sodium dodecyl sulfate, \(\upkappa \)-casein aggregation is devoid of a lag-phase and yields large oligomers that do not convert into amyloid fibrils.

Pseudo-one-dimensional nucleation in dilute polymer solutions.

Pathogenic protein fibrils have been shown in vitro to have nucleation-dependent kinetics despite the fact that one-dimensional structures do not have the size-dependent surface energy responsible for the lag time in classical theory. We present a theory showing that the conformational entropy of the peptide chains creates a free-energy barrier that is analogous to the translational entropy barrier in higher dimensions. We find that the dynamics of polymer rearrangement make it very unlikely for nucleation to succeed along the lowest free-energy trajectory, meaning that most of the nucleation flux avoids the free-energy saddle point. We use these results to construct a three-dimensional model for amyloid nucleation that accounts for conformational entropy, backbone H bonds, and side-chain interactions to compute nucleation rates as a function of concentration.

Fibril Growth Kinetics Reveal a Region of β2-microglobulin Important for Nucleation and Elongation of Aggregation

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β2-microglobulin (β2m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β2m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62–70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β2m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29–51 and 58–79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β2m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62–70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.

Nucleation of protein fibrillation by nanoparticles.

Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human beta(2)-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and beta(2)-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.

Triggers of Full-Length Tau Aggregation: A Role for Partially Folded Intermediates

Alzheimer's disease is characterized in part by the accumulation of full-length tau proteins into intracellular filamentous inclusions. To clarify the events that trigger lesion formation, the aggregation of recombinant full-length four-repeat tau (htau40) was examined in vitro under near-physiological conditions using transmission electron microscopy and spectroscopy methods. In the absence of exogenous inducers, tau protein behaved as an assembly-incompetent monomer with little tertiary structure. The addition of anionic inducers led to fibrillization with nucleation-dependent kinetics. On the basis of circular dichroism spectroscopy and reactivity with thioflavin S and 8-anilino-1-naphthalenesulfonic acid fluorescent probes, the inducer stabilized a monomeric species with the folding characteristics of a premolten globule state. Planar aromatic dyes capable of binding the intermediate state with high affinity were also capable of triggering fibrillization in the absence of other inducers. Dye-mediated aggregation was characterized by concentration-dependent decreases in lag time, indicating increased nucleation rates, and submicromolar critical concentrations, indicating a final equilibrium that favored the filamentous state. The data suggest that the rate-limiting barrier for filament formation from full-length tau is conformational and that the aggregation reaction is triggered by environmental conditions that stabilize assembly-competent conformations.

Amyloidogenesis recapitulated in cell culture: a peptide inhibitor provides direct evidence for the role of heparan sulfate and suggests a new treatment strategy.

To date 22 different polypeptides, including Abeta in Alzheimer's disease and PrP(Sc) in prion disorders, are known to re-fold and assemble into highly organized fibrils, which associate with heparan sulfate (HS) proteoglycans to form tissue deposits called amyloid. Mononuclear phagocytes have long been thought to be involved in this process, and we describe a monocytic cell culture system that can transform the acute-phase protein serum amyloid A (SAA1.1) into AA-amyloid and appears to recapitulate all the main features of amyloidogenesis observed in vivo. These features in common include nucleation-dependent kinetics, identical proteolytic processing of SAA1.1, and co-deposition of HS with the fibrils. Heparin and polyvinylsulfonate previously reported to block AA-amyloidogenesis in mice are also effective inhibitors in this cell culture model. Furthermore, a synthetic peptide (27-mer) corresponding to a HS binding site of SAA, blocks amyloid deposition at a concentration that is several-orders-of-magnitude lower than any other peptide-based inhibitor previously reported. The 27-mer's inhibitory activity may target the amyloidogenic pathway specifically as it does not interfere with the binding of SAA to monocytes. These data provide direct evidence that SAA1.1:HS interactions are a critical step in AA-amyloidogenesis and suggest a novel treatment strategy for other amyloidoses.