Biomedical subjects

Regina M Murphy

Publications and source records attributed to Regina M Murphy.

12 recordsLinked to original sources

PubMed2006

Kinetics of inhibition of beta-amyloid aggregation by transthyretin.

Deposition of beta-amyloid (Abeta) fibrils is an early event in the neurodegenerative processes associated with Alzheimer's disease. According to the "amyloid cascade" hypothesis, Abeta aggregation, and its subsequent deposition as fibrils, is the underlying cause of disease. Abeta is a proteolytic product of amyloid precursor protein (APP); several mutations in APP have been identified that are associated with early onset of disease. Transgenic mice overexpressing APP with the Swedish mutation develop numerous plaques but, surprisingly, lack the neurofibrillary tangles and neuronal loss characteristic of Alzheimer's disease, in apparent contradiction of the amyloid cascade hypothesis. However, recent studies suggest that coproduction of sAPPalpha, an alternative proteolytic product of APP, increases synthesis of transthyretin that, in turn, interacts directly with Abeta to inhibit its toxicity. Here we report results from biophysical analysis of Abeta aggregation kinetics in the presence of transthryetin. At substoichiometric ratios, transthyretin drastically decreased the rate of aggregation without affecting the fraction of Abeta in the aggregate pool. Detailed analysis of the data using a mathematical model demonstrated that the decrease in aggregation rate was due to both a decrease in the rate of elongation relative to the rate of initiation of filaments and a decrease in lateral association of filaments to fibrils. Tryptophan quenching data indicated that transthyretin binds weakly to Abeta, with an estimated apparent KS of 2300 M-1. Taken together, the data support a hypothesis wherein transthyretin preferentially binds to aggregated rather than monomeric Abeta and arrests further growth of the aggregates.

Amyloid beta-Peptides↗

PubMed2006

Phage display affords peptides that modulate beta-amyloid aggregation.

As the population ages, the need to develop methods to understand and intercept the processes responsible for protein aggregation diseases is becoming more urgent. The aggregation of the protein beta-amyloid (Abeta) has been implicated in Alzheimer's Disease (AD); however, whether the toxic species is a large, insoluble aggregate or some lower order form is not yet known. Agents that can modulate the aggregation state of Abeta could resolve this controversy by facilitating our understanding of the consequences of aggregation and its underlying mechanism. To date, however, ligands that bind to specific forms of Abeta have not been identified. To address this deficiency, we tested whether phage display could yield such ligands by screening libraries against Abeta in two different states: monomeric or highly aggregated. Intriguingly, the peptides selected had different effects on Abeta aggregation. Peptides selected for binding to monomeric Abeta did not perturb aggregation, but those selected using highly aggregated Abeta increase the rate of aggregation drastically. The latter also alter the morphology of the resulting aggregate. The ability of a peptide to promote aggregation correlated with its affinity for the N-terminal 10 residues of Abeta. This result indicates that the mechanism by which the peptides influence aggregation is related to their affinity for the Abeta N-terminus. Thus, the identification of compounds that bind to this Abeta section can afford agents that affect aggregation. Moreover, the data suggest that endogenous ligands that interact with the N-terminal region can influence the propensity of Abeta to form aggregates and the morphology of those that form. Our data highlight the utility of phage display for identifying ligands that bind to target proteins in different states, and they indicate that such agents can be used to perturb protein aggregation.

Amino Acid Sequence↗

PubMed2006

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 > or =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.

Amino Acid Sequence↗

PubMed2005

Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution.

The biophysical characterization of nonfunctional protein aggregates at physiologically relevant temperatures is much needed to gain deeper insights into the kinetic and thermodynamic relationships between protein folding and misfolding. Dynamic and static laser light scattering have been employed for the detection and detailed characterization of apomyoglobin (apoMb) soluble aggregates populated at room temperature upon dissolving the purified protein in buffer at pH 6.0, both in the presence and absence of high concentrations of urea. Unlike the beta-sheet self-associated aggregates previously reported for this protein at high temperatures, the soluble aggregates detected here have either alpha-helical or random coil secondary structure, depending on solvent and solution conditions. Hydrodynamic diameters range from 80 to 130 nm, with semiflexible chain-like morphology. The combined use of low pH and high urea concentration leads to structural unfolding and complete elimination of the large aggregates. Even upon starting from this virtually monomeric unfolded state, however, protein refolding leads to the formation of severely self-associated species with native-like secondary structure. Under these conditions, kinetic apoMb refolding proceeds via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily self-associated state bearing native-like secondary structure.

Animals↗

PubMed2005

Design of peptidyl compounds that affect beta-amyloid aggregation: importance of surface tension and context.

Self-association of beta-amyloid (Abeta) peptide into cross-beta-sheet fibrils induces cellular toxicity in vitro and is linked with progression of Alzheimer's disease. Previously, we demonstrated that hybrid peptides, containing a recognition domain that binds to Abeta and a disrupting domain consisting of a chain of charged amino acids, inhibited Abeta-associated toxicity in vitro and increased the rate of Abeta aggregation. In this work we examine the design parameter space of the disrupting domain. Using KLVFFKKKKKK as a base case, we tested hybrid compounds with a branched rather than linear lysine oligomer, with l-lysine replaced by d-lysine, and with lysine replaced by diaminopropionic acid. We synthesized a compound with a novel anionic disrupting domain that contained cysteine thiols oxidized to sulfates, as well as other compounds in which alkyl or ether chains were appended to KLVFF. In all cases, the hybrid compound's ability to increase solvent surface tension was the strongest predictor of its effect on Abeta aggregation kinetics. Finally, we investigated the effects of arginine on Abeta aggregation. Arginine is a well-known chaotrope but increases surface tension of water. Arginine modestly decreased Abeta aggregation. In contrast, RRRRRR slightly, and KLVFFRRRRRR greatly, increased Abeta aggregation. Thus, the influence of arginine on Abeta aggregation depends strongly on the context in which it is presented. The effect of arginine, RRRRRR, and KLVFFRRRRRR on Abeta aggregation was examined in detail using laser light scattering, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence, and transmission electron microscopy.

Amino Acid Sequence↗

PubMed2004

Urea modulation of beta-amyloid fibril growth: experimental studies and kinetic models.

Aggregation of beta-amyloid (Abeta) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Abeta, recent evidence supports the hypothesis that Abeta oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Abeta aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, beta-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Abeta aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.

Amyloid beta-Peptides↗

PubMed2004

Mechanism of accelerated assembly of beta-amyloid filaments into fibrils by KLVFFK(6).

Extracellular senile plaques are a central pathological feature of Alzheimer's disease. At the core of these plaques are fibrillar deposits of beta-amyloid peptide (Abeta). In vitro, Abeta spontaneously assembles into amyloid fibrils of cross-beta sheet structure. Although it was once believed that the fibrils themselves were toxic, more recent data supports the hypothesis that aggregation intermediates, rather than fully formed fibrils, are the most damaging to neuronal tissue. In previously published work, we identified several small peptides that interact with Abeta and increase its aggregation rate while decreasing its toxicity. In this work, we examined in detail the interaction between Abeta and one of these peptides. Using a mathematical model of Abeta aggregation kinetics, we show that the dominant effect of the peptide is to accelerate lateral association of Abeta filaments into fibrils.

Amyloid↗

PubMed2003

Kinetics of adsorption of beta-amyloid peptide Abeta(1-40) to lipid bilayers.

The Alzheimer's disease-related peptide beta-amyloid (Abeta) 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 Abeta 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) Abeta to lipid bilayers. In one method, the kinetics of Abeta adsorption and desorption to liposomes deposited onto a dextran-coated surface was measured using surface plasmon resonance. In the other method, Abeta was contacted with liposome-coated magnetic beads; adsorbed Abeta was separated from solution-phase peptide by use of a magnetic field. Monomeric Abeta adsorbed quickly but reversibly to lipid bilayers with low affinity, while aggregated Abeta 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, Abeta 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 Abeta toxicity, and in the search for inhibitors of toxicity.

Adsorption↗

PubMed2003

Targeted control of kinetics of beta-amyloid self-association by surface tension-modifying peptides.

Brain tissue from Alzheimer's patients contains extracellular senile plaques composed primarily of deposits of fibrillar aggregates of beta-amyloid peptide. beta-Amyloid aggregation is postulated to be a major factor in the onset of this neurodegenerative disease. Recently proposed is the hypothesis that oligomeric intermediates, rather than fully formed insoluble fibrils, are cytotoxic. Previously, we reported the discovery of peptides that accelerate beta-amyloid aggregation yet inhibit toxicity in vitro, in support of this hypothesis. These peptides contain two domains: a recognition element designed to bind to beta-amyloid and a disrupting element that alters beta-amyloid aggregation kinetics. Here we show that the aggregation rate-enhancing activity of the disrupting element correlates strongly with its ability to increase surface tension of aqueous solutions. Using the Hofmeister series as a guide, we designed a novel peptide with terminal side-chain trimethylammonium groups in the disrupting domain. The derivatized peptide greatly increased solvent surface tension and accelerated beta-amyloid aggregation kinetics by severalfold. Equivalent increases in surface tension in the absence of a recognition domain had no effect on beta-amyloid aggregation. These results suggest a novel strategy for targeting localized changes in interfacial energy to specific proteins, as a way to selectively alter protein folding, stability, and aggregation.

Amyloid beta-Peptides↗

PubMed2002

Affinity-based inhibition of beta-amyloid toxicity.

Strategies for interfering with protein aggregation are important for elucidating and controlling the pathologies of amyloid diseases. We have previously identified compounds that block the cellular toxicity of the beta-amyloid peptide, but the relationship between their ability to inhibit toxicity and their affinity for A beta is unknown. To elucidate this relationship, we have developed an assay capable of measuring the affinities of small molecules for beta-amyloid peptide. Our approach employs immobilized beta-amyloid peptide at low density to minimize the problems that arise from variability in the beta-amyloid aggregation state. We found that low-molecular weight (MW of 700-1700) ligands for beta-amyloid can be identified readily by using surface plasmon resonance. The best of these bound effectively (K(d) approximately 40 microM) to beta-amyloid. The affinities measured for peptides in the SPR assay correspond to results from A beta cell toxicity assays. The most potent ligands for immobilized beta-amyloid are the most potent inhibitors of the neuronal cell toxicity of beta-amyloid. Compounds with dissociation constants above approximately 100 microM did not show significant activity in the cell toxicity assays. Our data support the hypothesis that ligands exhibiting greater affinity for the beta-amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity.

Amino Acid Sequence↗

PubMed2002

Peptide aggregation in neurodegenerative disease.

In the not-so-distant past, insoluble aggregated protein was considered as uninteresting and bothersome as yesterday's trash. More recently, protein aggregates have enjoyed considerable scientific interest, as it has become clear that these aggregates play key roles in many diseases. In this review, we focus attention on three polypeptides: beta-amyloid, prion, and huntingtin, which are linked to three feared neurodegenerative diseases: Alzheimer's, "mad cow," and Huntington's disease, respectively. These proteins lack any significant primary sequence homology, yet their aggregates possess very similar features, specifically, high beta-sheet content, fibrillar morphology, relative insolubility, and protease resistance. Because the aggregates are noncrystalline, secrets of their structure at nanometer resolution are only slowly yielding to X-ray diffraction, solid-state NMR, and other techniques. Besides structure, the aggregates may possess similar pathways of assembly. Two alternative assembly pathways have been proposed: the nucleation-elongation and the template-assisted mode. These two modes may be complementary, not mutually exclusive. Strategies for interfering with aggregation, which may provide novel therapeutic approaches, are under development. The structural similarities between protein aggregates of dissimilar origin suggest that therapeutic strategies successful against one disease may have broad utility in others.

Amino Acid Sequence↗

PubMedDate not supplied

Predicting solvent and aggregation effects of peptides using group contribution calculations.

Assembly of proteins and peptides into amyloid fibrils is characteristic of the pathology of a number of degenerative diseases, of which perhaps the best known is Alzheimer's. Neuronal death in Alzheimer's disease has been linked to aggregation of beta-amyloid peptide and deposition of the aggregates in brain tissue. Recent studies suggest that soluble intermediate oligomers rather than fully formed fibrils may be responsible for beta-amyloid's toxicity; consistent with this hypothesis, some compounds that inhibited beta-amyloid toxicity were found to accelerate aggregation of the peptide. The degree of increase of aggregation caused by these compounds was shown to correlate strongly with their effect on the surface tension of aqueous buffers (Gibson, T. J.; Murphy, R. M. Biochemistry 2005, 44, 8898-8907). In work reported here, we used group contribution methods to calculate infinite dilution activity coefficients of moieties contained in these compounds. We demonstrate that activity coefficients calculated in this manner are strongly predictive of effects of these peptides on solvent surface tension and on beta-amyloid aggregation. These results suggest that group contribution calculations can be used for a priori design of compounds that modulate aggregation of peptides through their effect on solvent properties.

Amino Acid Sequence↗