Regina M. Murphy

SOLVENT EFFECTS ON SELF-ASSEMBLY OF BETA -AMYLOID PEPTIDE

beta-amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimers disease patients. Synthetic A beta spontaneously assembles into amyloid fibrils and is neurotoxic to cortical cultures. Neurotoxicity has been associated with the degree of peptide aggregation, yet the mechanism of assembly of A beta into amyloid fibrils is poorly understood. In this work, A beta was dissolved in several different solvents commonly used in neurotoxicity assays. In pure dimethylsulfoxide (DMSO), A beta had no detectable beta-sheet content; in 0.1% trifluoroacetate, the peptide contained one-third beta-sheet; and in 35% acetonitrile/0.1% trifluoroacetate, A beta was two-thirds beta-sheet, equivalent to the fibrillar peptide in physiological buffer. Stock solutions of peptide were diluted into phosphate-buffered saline, and fibril growth was followed by static and dynamic light scattering. The growth rate was substantially faster when the peptide was predissolved in 35% acetonitrile/0.1% trifluoroacetate than in 0.1% trifluoroacetate, 10% DMSO, or 100% DMSO. Differences in growth rate were attributed to changes in the secondary structure of the peptide in the stock solvent. These results suggest that formation of an intermediate with a high beta-sheet content is a controlling step in A beta self-assembly.

A strategy for designing inhibitors of beta-amyloid toxicity.

β-Amyloid peptide is the major protein component of Alzheimers plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of β-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with β-amyloid, is combined with a disrupting element, which alters β-amyloid aggregation pathways. The synthesis, biophysical characterization, and biological activity of such an inhibitor is reported. This prototype inhibitor is composed of residues 15-25 of β-amyloid peptide, designed to function as the recognition element, linked to an oligolysine disrupting element. The inhibitor does not alter the apparent secondary structure of β-amyloid nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. Evidence for these effects includes changes in fibril morphology and a reduction in thioflavin T fluorescence. In addition to its influence on the physical properties of β-amyloid aggregates, the inhibitor completely blocks β-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of β-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.

Kinetics of aggregation of synthetic β-amyloid peptide

Abstract β-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimers disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-β conformation, β-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of β-amyloid peptide, β(1–40), was synthesized and characterized. β(1–40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that, β(1–40) contained approximately two-thirds β-structure, but no α-helical character. Quasielastic and classical light scattering measurements showed that β(1–40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 × 106 after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.

Beta-amyloid peptide blocks the fast-inactivating K+ current in rat hippocampal neurons

Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.

Examining Polyglutamine Peptide Length: A Connection between Collapsed Conformations and Increased Aggregation

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntingtons. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from approximately 11 to approximately 7 A as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 degrees C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.

Light scattering analysis of fibril growth from the amino-terminal fragment beta(1-28) of beta-amyloid peptide.

beta-Amyloid protein (beta-A/4) is the major protein component of Alzheimer disease-related senile plaques and has been postulated to be a significant contributing factor in the onset and/or progression of the disease. In the senile plaque, beta-A/4 appears as bundles of amyloid fibrils. The biological activity of beta-A/4 may be related to its state of aggregation. In this work, self-assembly, fibril formation, and interfibrillary aggregation of beta(1-28), a synthetic peptide homologous with the amino-terminal fragment of beta-A/4, were investigated. The predominant form of beta(1-28) detected by size-exclusion chromatography and polyacrylamide gel electrophoresis was apparently a tetramer which does not bind Congo red. Aggregates containing cross-beta sheet structures which bind Congo red and thioflavin T were observed at concentrations of approximately 0.3 mg/ml or greater. Concentrations of 0.5-1 mg/ml were necessary for aggregation into fibrils to be detectable by classical or quasielastic light scattering. Both fibril elongation and fibril-fibril aggregation occur over the time scale investigated. The kinetics of aggregation were much faster at physiological salt concentrations than at lower ionic strength. Ionic strength also appeared to influence the morphology of the fibril aggregates. The data indicate that sample preparation method and sample history influence fibril size and number density.

Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide

beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimers patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of ≥100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12–17 of full‐length insulin, was identified as a short peptide that interacts with full‐length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near‐equimolar concentrations. An effective binding domain and N‐terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two‐step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.

Kinetics of adsorption of β-amyloid peptide Aβ(1–40) to lipid bilayers

The Alzheimers disease-related peptide β-amyloid (Aβ) is toxic to neurons. The toxicity of the peptide appears to require conversion of the monomeric form to an aggregated fibrillar species. The interaction of Aβ with cell membranes has attracted interest as one plausible mechanism by which the peptide exerts its toxic activity. We developed two methods to measure the adsorption of fresh (monomeric) and aged (aggregated) Aβ to lipid bilayers. In one method, the kinetics of Aβ adsorption and desorption to liposomes deposited onto a dextran-coated surface was measured using surface plasmon resonance. In the other method, Aβ was contacted with liposome-coated magnetic beads; adsorbed Aβ was separated from solution-phase peptide by use of a magnetic field. Monomeric Aβ adsorbed quickly but reversibly to lipid bilayers with low affinity, while aggregated Aβ adsorbed slowly but irreversibly. These two methods provide complementary means of quantifying the adsorption of aggregating proteins to membranes. The results correlate strongly with previous observations that fibrillar, but not monomeric, Aβ restricts the motion of acyl tails in phospholipid bilayers. The methods should be useful for further elucidation of the role of membrane adsorption in mediating Aβ toxicity, and in the search for inhibitors of toxicity.

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 beta-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.